CN103739323B - A kind of preparation method of composite bio-fertilizer - Google Patents
A kind of preparation method of composite bio-fertilizer Download PDFInfo
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- CN103739323B CN103739323B CN201310743961.7A CN201310743961A CN103739323B CN 103739323 B CN103739323 B CN 103739323B CN 201310743961 A CN201310743961 A CN 201310743961A CN 103739323 B CN103739323 B CN 103739323B
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- 239000003337 fertilizer Substances 0.000 title claims abstract description 32
- 238000002360 preparation method Methods 0.000 title claims abstract description 28
- 239000002131 composite material Substances 0.000 title claims abstract description 26
- 241000228245 Aspergillus niger Species 0.000 claims abstract description 26
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims abstract description 15
- 229910052698 phosphorus Inorganic materials 0.000 claims abstract description 15
- 239000011574 phosphorus Substances 0.000 claims abstract description 15
- 244000063299 Bacillus subtilis Species 0.000 claims abstract description 14
- 235000014469 Bacillus subtilis Nutrition 0.000 claims abstract description 13
- 241000194107 Bacillus megaterium Species 0.000 claims abstract description 12
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 5
- 230000000813 microbial effect Effects 0.000 claims abstract description 5
- 238000000855 fermentation Methods 0.000 claims description 22
- 230000004151 fermentation Effects 0.000 claims description 22
- 239000000463 material Substances 0.000 claims description 22
- 241000894006 Bacteria Species 0.000 claims description 21
- 230000001580 bacterial effect Effects 0.000 claims description 21
- 239000007787 solid Substances 0.000 claims description 17
- 239000002609 medium Substances 0.000 claims description 16
- 238000001035 drying Methods 0.000 claims description 15
- 239000007788 liquid Substances 0.000 claims description 15
- 239000002054 inoculum Substances 0.000 claims description 12
- 238000011534 incubation Methods 0.000 claims description 11
- 230000001954 sterilising effect Effects 0.000 claims description 11
- 238000011218 seed culture Methods 0.000 claims description 10
- 241000196324 Embryophyta Species 0.000 claims description 9
- 238000011068 loading method Methods 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 8
- 238000003756 stirring Methods 0.000 claims description 8
- 239000001963 growth medium Substances 0.000 claims description 6
- 239000002068 microbial inoculum Substances 0.000 claims description 6
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- 235000020679 tap water Nutrition 0.000 claims description 6
- 239000000843 powder Substances 0.000 claims description 5
- 238000009423 ventilation Methods 0.000 claims description 5
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- 108010080698 Peptones Proteins 0.000 claims description 4
- 235000019319 peptone Nutrition 0.000 claims description 4
- 239000011343 solid material Substances 0.000 claims description 4
- 238000010563 solid-state fermentation Methods 0.000 claims description 4
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- 239000004375 Dextrin Substances 0.000 claims description 3
- 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 claims description 3
- 244000068988 Glycine max Species 0.000 claims description 3
- 235000010469 Glycine max Nutrition 0.000 claims description 3
- 230000004913 activation Effects 0.000 claims description 3
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- 238000003892 spreading Methods 0.000 claims description 3
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- 238000012136 culture method Methods 0.000 claims description 2
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- 239000000047 product Substances 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
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- 102000004139 alpha-Amylases Human genes 0.000 description 7
- 108090000637 alpha-Amylases Proteins 0.000 description 7
- 238000010790 dilution Methods 0.000 description 7
- 239000012895 dilution Substances 0.000 description 7
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- 239000002689 soil Substances 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
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- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
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- 239000011591 potassium Substances 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
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- 229940024171 alpha-amylase Drugs 0.000 description 4
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- 229910019142 PO4 Inorganic materials 0.000 description 3
- 240000008042 Zea mays Species 0.000 description 3
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 3
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 3
- 235000011130 ammonium sulphate Nutrition 0.000 description 3
- 235000013339 cereals Nutrition 0.000 description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 3
- 235000019341 magnesium sulphate Nutrition 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 3
- 239000010452 phosphate Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
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- 241000209140 Triticum Species 0.000 description 2
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- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 2
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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 1
- 244000153158 Ammi visnaga Species 0.000 description 1
- 235000010585 Ammi visnaga Nutrition 0.000 description 1
- 101710130006 Beta-glucanase 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
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- KIWBPDUYBMNFTB-UHFFFAOYSA-N Ethyl hydrogen sulfate Chemical compound CCOS(O)(=O)=O KIWBPDUYBMNFTB-UHFFFAOYSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
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- 229930195725 Mannitol Natural products 0.000 description 1
- 108090000913 Nitrate Reductases Proteins 0.000 description 1
- 241000237502 Ostreidae Species 0.000 description 1
- 102000015439 Phospholipases Human genes 0.000 description 1
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- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 241001148470 aerobic bacillus Species 0.000 description 1
- SRBFZHDQGSBBOR-QMKXCQHVSA-N alpha-L-arabinopyranose Chemical compound O[C@H]1CO[C@@H](O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-QMKXCQHVSA-N 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
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- IQFVPQOLBLOTPF-HKXUKFGYSA-L congo red Chemical compound [Na+].[Na+].C1=CC=CC2=C(N)C(/N=N/C3=CC=C(C=C3)C3=CC=C(C=C3)/N=N/C3=C(C4=CC=CC=C4C(=C3)S([O-])(=O)=O)N)=CC(S([O-])(=O)=O)=C21 IQFVPQOLBLOTPF-HKXUKFGYSA-L 0.000 description 1
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- 238000011081 inoculation Methods 0.000 description 1
- 239000010871 livestock manure Substances 0.000 description 1
- 235000009973 maize Nutrition 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of preparation method of high-efficiency composite biofertilizer, be specially: by aspergillus niger culture 20-50 part of following parts by weight, phosphorus decomposing bacillus megaterium 5-10 part, bacillus subtilis microbial agent 10-30 part, carry out respectively cultivating, fermenting, then tunning is combined as high-efficiency composite biofertilizer in proportion.Experiment shows, uses the product that obtains of the present invention to improve grain yield 8-30%, improves vegetable crop 10-40%, also can improve vegetables, the quality of grain and local flavor.The amount of every mu of land use fertilizer of the present invention is 50-100 gram, is one of high-efficiency composite biofertilizer that usage quantity is less both at home and abroad at present.
Description
Technical field:
Invent the fertilizer related in agrotechnique, particularly a kind of preparation method of novel composite bio-fertilizer.
Background technology:
Existing bio-feritlizer is monistic bio-feritlizer mostly.As: azotobacteria fertilizer, primarily of bacterium and the fungi composition with nitrogen fixation; And for example: phosphorus bacteria, its function decomposes the organophosphorus in soil, makes it to become the phosphorus element being easy to be absorbed by plants; For another example: bacterium, its function decomposes the potassium compound in soil, makes it to become the available potassium that can be absorbed and used by plants.These fertilizer play each self-applying being used alone Shi Junneng, and part meets plant to nitrogen, phosphorus, the needs of potassium three elements.They can not produce the soil compaction phenomenon that life-time service chemical fertilizer causes.But its deficiency is: be used alone the aspect that above-mentioned three class bio-feritlizers can only solve plant desired nutritional, and cost is higher, consumption is large, meanwhile, by three with the use of, be difficult to again deficiency or waste that the ratio of accomplishing suitably causes rate of fertilizer application.Facts have proved of the bio-feritlizer of the single or compound of life-time service, they can only replacing fertilizer less than 30%.
Summary of the invention:
The object of the invention is the basic condition for China's soil fertility, a kind of preparation method of composite bio-fertilizer is provided.
For solving the problem, the technical solution adopted in the present invention is as follows:
A preparation method for composite bio-fertilizer, is specially and elite aspergillus niger culture, phosphorus decomposing bacillus megaterium, subtilis is carried out respectively cultivating, fermenting, then tunning is combined as composite bio-fertilizer in proportion.
Described composite bio-fertilizer, is made up of the raw material of following parts by weight: aspergillus niger culture 20-50 part, phosphorus decomposing bacillus megaterium 5-10 part, bacillus subtilis microbial agent 10-30 part;
Phosphorus decomposing bacillus megaterium is a kind in bacillus (Bacillus).Motion, forms pod membrane, aerobic.Acid is produced from glucose, also normal from pectinose and the acid of N.F,USP MANNITOL product.Hydrolyzed starch, does not produce lecithinase, and VP is negative.The organophosphorus that can decompose in soil becomes the available rapid available phosphorus of plant.Phosphorus decomposing bacillus megaterium microbial inoculum is provided by Cangzhou prosperous generation thing technical institute, address: liberation West Road, Hebei China Cangzhou City Canal Zone chin or cheek and Building A 1, international business affairs center district 807-812.
Prepared by aspergillus niger culture: spawn culture, adopts conventional solid fermentation culture method: spore liquid is inoculated in solid state fermentation culture material, and 26-35 DEG C is cultured to mycelia and covers with culture material, and low temperature fluidized bed dry, pulverize dry thing.
Beneficial effect:
Experiment shows, uses the product that obtains of the present invention to improve grain yield 8-30%, improves vegetable crop 10-40%, also can improve vegetables, the quality of grain and local flavor.The amount of every mu of land use fertilizer of the present invention is 60-100 gram, is one of composite bio-fertilizer that usage quantity is less both at home and abroad at present.
Every mu of usage quantity is 80-200 gram.In composite bio-fertilizer of the present invention, add the tap water of 120 times and the urea of 6 times, activation 28 hours in the water temperature of less than 45 DEG C more than 22 DEG C, then together with chemical fertilizer (according to last year usage quantity 50%) be spilled in soil.Preferably make base manure, annual use 1-3 time.
Embodiment:
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.1, preparation cultivated by microbial inoculum:
Preparation cultivated by bacillus subtilis microbial inoculum: cultivate subtilis from inclined-plane switching, the seed liquor after spreading cultivation step by step is transferred in fermentor tank, and the complete fermentation liquor low-temperature negative-pressure that ferments is concentrated in vacuo to the 40-50% of original volume, obtains bacterium concentrated solution.Add carrier: in concentrated solution, add the carrier mixed, mix; The weight ratio of concentrated solution and carrier is 0.5-0.7:1, and vehicle group becomes: CaCO
320-40 part, dextrin 10-20 part.Fluidised bed drying, drying temperature 50 DEG C.
Prepared by aspergillus niger culture: spawn culture, and solid fermentation is cultivated: spore liquid is inoculated in solid state fermentation culture material, and 26-35 DEG C is cultured to mycelia and covers with culture material, and low temperature fluidized bed dry, pulverize dry thing;
The bacterial strain of product Thermostable α-Amylase provided by the invention is specially subtilis (Bacillussubtilis) Li-2013-02.This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (be called for short CGMCC, address is: No. 3, No. 1, North Star West Road, Chaoyang District, city of BeiJing, China institute) on July 15th, 2013, and preserving number is CGMCCNo.7926.
Described bacterial strain feature is as follows:
Described bacterial strain colony colour on solid plate is oyster white, and surface drying is opaque, 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 the positive.
Described subtilis (Bacillussubtilis) Li-2013-02 is preserved in the product Thermostable α-Amylase in this laboratory subtilis Li-2013 by a strain obtains through UV-LiCl-ethyl sulfate Mutation screening, and concrete screening step is as follows:
(1) preparation of bacteria suspension
The mono-bacterium colony of Li-2013 grown after plate streaking is separated is accessed in seed culture medium, 100r/min, after 40 DEG C of cultivation 12h, after getting 1mL medium centrifugal, use brine twice, and resuspended with 9mL physiological saline.
(2) UV-LiCl-ethyl sulfate complex mutation
Bacteria suspension is placed in aseptic flat board, is 30cm in distance, stirs and irradiate 100s under the ultraviolet lamp of power 15w.Bacterium liquid through irradiating is coated lithium chloride flat board after gradient dilution, and contrasts to be coated with flat board without the bacterium liquid dilution of uv irradiating.Above-mentioned coating is dull and stereotyped uniformly, wrap with the cloth of black or newspaper, put 40 DEG C and cultivate 48h, the flat board growing bacterium colony filters out hydrolysis circle choose preserve to inclined-plane with colony diameter ratio the maximum, bacteria suspension is mixed with after purifying, fully mix with ethyl sulfate stoste after gradient dilution, and in 40 DEG C of concussion process 40min, the bacterium liquid processed is coated lithium chloride flat board after gradient dilution.
(3) primary dcreening operation of high-yield strains
Above-mentioned coating is dull and stereotyped uniformly, and put 40 DEG C and cultivate 48h, on the flat board growing bacterium colony, primary dcreening operation goes out hydrolysis circle and chooses preserve to inclined-plane with colony diameter ratio the greater, obtains three strain bacterium Li-2013-01, Li-2013-02, Li-2013-03 after purifying
(4) shake flask fermentation sieves again
By the three strain bacterium Li-2013-01 obtained, Li-2013-02, Li-2013-03 carry out shake flask fermentation in the 250mL shaking flask containing 30mL fermention medium, seed inoculum size 10% (V/V), 40 DEG C, 100r/min cultivates 72h, centrifuging and taking fermented supernatant fluid obtains crude enzyme liquid.
(5) enzyme activity determination
The definition of Mei Huo unit: 1mL crude enzyme liquid, in 105 DEG C, under pH4.2 condition, 1min liquefies 1mg Zulkovsky starch, is 1 enzyme activity unit, represents with U/mL.
After measured, bacterial strain Li-2013-02 is stable most superior strain, and enzyme is lived and reached 30000U/mL, to live raising 1.6 times than original strain enzyme.
Described lithium chloride is dull and stereotyped: starch 1%, peptone 1%, (NH
3)
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%, NaCl1%.
Described fermention medium: Semen Maydis powder 5% ~ 15%, soybean cake powder 4% ~ 10%, (NH
3)
2sO
40.4%, K
2hPO
40.8%, CaCl
20.2%.
Described shake flask culture conditions: this bacterium in the 250mL shaking flask containing 30mL fermention medium, inoculum size 10% (V/V), 100r/min, 40 DEG C of fermentation culture 72h.
Fermented by bacterial strain Li-2013-02 and obtain a kind of high-temperature resistant alpha-amylase, its zymologic property is as follows:
(1) this enzyme thermal adaptation a wider range, optimum temperature is between 105-115 DEG C, and the temperature stability of preserving below 110 DEG C is better, and more than 115 DEG C to preserve long-time temperature stability poor.
(2) this enzyme optimal reaction pH value is 4.2.High enzyme vigor is all had, the enzyme complete stability alive when pH value is 3.0 between pH value 3.0-7.0.
(3) enzymic activity: by mutant strain Li-2013-02 provided by the present invention, the Thermostable α-Amylase enzyme activity of preparation is 30000-35000U/ml.
1, the present invention uses the method for UV-LiCl-ethyl sulfate complex mutation to obtain the subtilis Li-2013-02 of a plant height product Thermostable α-Amylase, and this bacterial strain has acidproof, thermotolerance by force, produces the feature that enzyme activity is high.
2, this bacterial strain is had to produce the Thermostable α-Amylase enzyme activity of gained up to 30000-35000u/ml; Applicable temperature scope is 25-115 DEG C, optimal reactive temperature 110 DEG C, at 110 DEG C of enzymes complete stability alive; Being suitable for pH value in reaction scope is 3.0-7.0, the enzyme complete stability alive when pH value is 3.0, optimal reaction pH value is 4.2, higher than existing Thermostable α-Amylase enzyme activity, enzyme effect optimum pH wide scope, resistance to temperature is high, is particularly suitable for that temperature of reaction is high, liquefaction process and Mashing process the industrialization demand of depositing.
Aspergillus niger strain AspergillusnigerLi-2013-03 provided by the invention is through taking turns nitrosoguanidine mutagenesis by the aspergillus niger AspergillusnigerLi-2010 of a strain cellulase-producing of Laboratories Accession more, then mutant strain step-sizing is eliminated, finally through leavening property test screen, the Aspergillus niger strain AspergillusnigerLi-2013-03 producing high activity cellulase is obtained to strain excellent.
The bacterial strain of the high activity cellulase of product provided by the invention is specially aspergillus niger AspergillusnigerLi-2013-03.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, city of BeiJing, China institute, postcode 100101), preserving number is CGMCCNO.7927.
Produce the screening method of high activity cellulase bacterial strain, comprise the following steps:
1) slant culture: by original Aspergillus niger strain AspergillusnigerLi-2010 streak inoculation slant medium, 30 DEG C cultivate 2 ~ 3d, until mycelium ripe, produce a large amount of black spore.Described slant medium is composed as follows: 12 ° of Brix wort l000mL, pH value nature, 121 DEG C of sterilizing 20min;
2) spore suspension preparation (following steps all aseptically operate): add 15mL sterilized water to test tube slant, spore is scraped, with filter paper filtering, filtered solution poured into sterilizing and be added with in the 150mL triangular flask of 5-10 grain sterile glass beads, triangular flask is put into shaking table vibration 10-15min, spore is disperseed.
3) nitrosoguanidine (NTG) mutagenesis
A. with sterilized water spore suspension is adjusted to and is diluted to 10
6-10
7individual/mL.
B. get 10mL bacteria suspension to be transferred in 100mL triangular flask, add the NTG of 10mg, be mixed with the NTG solution that final concentration is 10mg/mL, and add 4-5 and drip acetone, be beneficial to NTG and dissolve.
C. at 30 DEG C, the centrifugal 10min of 200rpm oscillatory reaction 30min, 5000rpm collects thalline, with stroke-physiological saline solution washing several, and stopped reaction.
D. suitably spore concentration is adjusted to 10 by dilution
3individual/mL, get last dilution bacterium liquid 0.2mL, dilution spread is on Mierocrystalline cellulose-Congo red plate screening substratum.The bacterial strain 200 that after cultivating 2 ~ 3 days at 30 DEG C, picking transparent circle/colony diameter is larger.Described Mierocrystalline cellulose-Congo red plate screening substratum is composed as follows: cellulose powder 10g, Congo red 0.2g, ammonium sulfate 5g, magnesium sulfate 0.25g, potassium primary phosphate 1g, sodium-chlor 0.1g, gelatin 2g, agar 20g, tap water constant volume 1000mL, pH value 5-6,121 DEG C of sterilizing 20min.
E. sieve again: the 200 strain bacterium obtained are inoculated in slant medium with sterile toothpick respectively, and 30 DEG C are cultured to spore and are paved with inclined-plane.Respectively spore is equipped with 50mL and sieves again in the 250mL triangular flask of substratum ferment to be inoculated under aseptic washing, inoculum size 10% (v/v), 30 DEG C, 100r/min cultivates 96h, measures the cellulase activity of each bacterial strain respectively.The described substratum of sieve is again composed as follows: cellulose powder 50g, ammonium sulfate 5g, magnesium sulfate 0.25g, potassium primary phosphate 1g, sodium-chlor 0.1g, tap water constant volume 1000mL, pH value 5-6,121 DEG C of sterilizing 20min.The bacterial strain choosing cellulose enzyme vigor the highest carries out amplification test.
4) genetic stability test
Li-2013-03 bacterial strain is gone down to posterity for continuous ten times on inclined-plane, and detects the fermentation situation after at every turn going down to posterity by the method that shaking flask is sieved again.Experiment finds, inclined-plane goes down to posterity for continuous ten times, 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.
5) scale-up
1. seed culture: strains A spergillusnigerLi-2013-03 the highest for cellulose enzyme vigor is accessed in 500mL triangular flask, seed culture medium loading amount 100 milliliters, 30 DEG C, 150rpm shaking table cultivation 72-96h.
3. seed tank culture: seed liquor is equipped with in the 10L fermentor tank of 7.5L fermented liquid with the access of 10% (v/v) inoculum size, control ph is constant is 6.0 ± 0.2, culture temperature 30 ± 0.1 DEG C, stirring velocity 300rpm, ventilation (v/v) 1:0.8-1.2, incubation time 96h, dissolved oxygen 20-30%.Described fermention medium consists of: cellulose powder 100g, ammonium sulfate 5g, magnesium sulfate 0.25g, potassium primary phosphate 1g, sodium-chlor 0.1g, tap water constant volume 1000mL, pH value 5-6,121 DEG C of sterilizing 20min.
After fermentation ends, get fermented supernatant fluid (crude enzyme liquid) and carry out enzyme activity detection after measured, the circumscribed beta-glucanase of strains A spergillusnigerLi-2013-03, Endo-β-glucanase, beta-glucosidase and filter paper enzyme activity reach 620U/mL, 1289U/mL, 456U/mL and 732U/mL respectively, improve 9.21 times, 7.43 times, 8.15 times and 10.31 times respectively than starting strain AspergillusnigerLi-2010.
The preparation method of embodiment 1 one kinds of composite bio-fertilizers, is specially and elite aspergillus niger culture, phosphorus decomposing bacillus megaterium, subtilis is carried out respectively cultivating, fermenting, then tunning is combined as composite bio-fertilizer in proportion;
Described composite bio-fertilizer, by the raw material of following parts by weight, composition: aspergillus niger culture 40 parts, phosphorus decomposing bacillus megaterium 8 parts, bacillus subtilis microbial agent 20 parts;
The preparation method of described composite bio-fertilizer comprises the steps:
Preparation cultivated by microbial inoculum:
Preparation cultivated by bacillus subtilis microbial inoculum: cultivate subtilis from inclined-plane switching, the seed liquor after spreading cultivation step by step is transferred in fermentor tank, and the complete fermentation liquor low-temperature negative-pressure that ferments is concentrated in vacuo to 50% of original volume, obtains bacterium concentrated solution.Add carrier: in concentrated solution, add the carrier mixed, mix; The weight ratio of concentrated solution and carrier is 0.6:1, and vehicle group becomes: CaCO
330 parts, 15 parts, dextrin.Fluidised bed drying, drying temperature 50 DEG C.
Prepared by aspergillus niger culture: spawn culture, and solid fermentation is cultivated: spore liquid is inoculated in solid state fermentation culture material, and 30 DEG C are cultured to mycelia and cover with culture material, and low temperature fluidized bed 45 DEG C dry, pulverize dry thing;
The bacterial classification adopted is as follows:
Subtilis (Bacillussubtilissubsp) CGMCC7926
The depositary institution of above-mentioned bacterial classification is China General Microbiological culture presevation administrative center.Address: No. 13, Zhongguancun N 1st Lane, Beijing City institute of microbiology of the Chinese Academy of Sciences; Postcode: 100080.
The preparation method of aspergillus niger culture:
Slant strains activation culture: be transferred on slant medium by aspergillus niger slant strains, cultivates 3 days for 27 DEG C.
Solid first order seed is cultivated: the access of picking aspergillus niger slant strains is equipped with in 500 milliliters of triangular flasks of 100 grams of substratum and is carried out seed culture, cultivates 3 days for 30 DEG C.
Solid secondary seed is cultivated: stirred by above-mentioned cultured solid first order seed and carry out seed culture, culture condition for adding in 5000 milliliters of triangular flasks that 1000 grams of substratum are housed after fragment: cultivate 3 days for 30 DEG C.
Solid fermentation is cultivated: pulverized by second-level shake flask seed, add in the fermentation vat or pallet that sterilising medium is housed and mix rear cultivation, and bent material culture temperature controls at 26-35 DEG C, humidity 80-90%, every 10 hours stirrings once, and incubation time 5-7 days; The cultivation of solid koji material adopts conventional bent material culture technique; Treat culture material cover with mycelia can terminate cultivate, substratum in advance through high temperature steaming sterilising treatment, sterilising conditions control temperature 121 DEG C, 1 hour time.
Drying and crushing: fermentation ends culture material carries out drying on fluidized-bed or other drying plants, drying temperature controls, at 60 DEG C, to be dried to moisture content below 10%, then to be pulverized by solid culture medium, and crushing material aperture is more than 60 orders.
Substratum forms: solid material adds equivalent tap water; Initial pH nature; Described solid material mass percent consists of: wheat bran 70%, the crop material 15% of pulverizing, soybean cake powder 5%, W-Gum 10%.
The preparation method of subtilis:
1. 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 accessed in 500 ml shake flasks, substratum loading amount 100 milliliters, rotary shaker 180 revs/min, culture temperature 30 DEG C, incubation time 24 hours;
(2) secondary seed cultivate: by first order seed according to 10% inoculum size access in 500 milliliters of secondary seed shaking flasks, 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 flask with 10% inoculum size, substratum loading amount 1000 milliliters, rotary shaker 100 revs/min, culture temperature 30 DEG C, incubation time 24 hours;
(4) first class seed pot is cultivated: the first class seed pot being 150L with 10% inoculum size access cubic capacity by three grades of seeds, fermention medium loading amount 100L, culture temperature 28 DEG C, stirring velocity 100 revs/min, 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 is accessed cubic capacity with 10% inoculum size, fermention medium loading amount 1 ton, culture condition culture temperature 28 DEG C, stirring velocity 100 revs/min, ventilation (V/V) 1:0.5, tank pressure 0.05Mpa, incubation time 24 hours.
Substratum forms: glucose 6%, yeast extract 1%, peptone 0.2%, CaCO
31%, pH6.8.
Selection experimental field and test design: test and carry out in Ba Bao village, dapple pond town, Yanchi county Ningxia 27 days-September 30 March in 2012.
The equal maize planting in experimental plot and contrast field 10 mu, test group uses product every mu of the present invention 0.1 kilogram when planting respectively, emerges about 1 month and uses invention product 0.7 kilogram by loose ground mode, and control group uses common fertilizer.
Use the corn field of product of the present invention, corn yield reaches 650 kilograms, and control group reaches 520 kilograms; This plot was at the 2nd year plant spring wheat, and spring wheat production reaches 410 kilograms, improves 20% than control group per unit area yield.And experimental plot Soil structure is good, without bulk with harden.
Claims (8)
1. a preparation method for composite bio-fertilizer, is characterized in that, elite aspergillus niger culture, phosphorus decomposing bacillus megaterium, subtilis is carried out respectively cultivating, fermenting, is then combined in proportion by tunning;
Described composite bio-fertilizer, is made up of the raw material of following parts by weight: aspergillus niger culture 20-50 part, phosphorus decomposing bacillus megaterium 5-10 part, bacillus subtilis microbial agent 10-30 part; Subtilis (Bacillussubtilis) Li-2013-02 preserving number is CGMCCNo.7926, aspergillus niger (Aspergillusniger) Li-2013-03 preserving number is CGMCCNO.7927, phosphorus decomposing bacillus megaterium (Bacillusmegaterium).
2. the preparation method of composite bio-fertilizer according to claim 1, it is characterized in that, the parts by weight of high-efficiency composite biofertilizer consist of aspergillus niger culture 40 parts, phosphorus decomposing bacillus megaterium 8 parts, bacillus subtilis microbial agent 20 parts.
3. the preparation method of composite bio-fertilizer according to claim 1, it is characterized in that, preparation method is as follows for bacillus subtilis microbial inoculum:
Cultivate subtilis from inclined-plane switching, the seed liquor after spreading cultivation step by step is transferred in fermentor tank, and the complete fermentation liquor low-temperature negative-pressure that ferments is concentrated in vacuo to the 40-50% of original volume, obtains bacterium concentrated solution; Add carrier: in concentrated solution, add the carrier mixed, mix; The weight ratio of concentrated solution and carrier is 0.5-0.7:1, and vehicle group becomes: CaCO
320-40 part, dextrin 10-20 part; Fluidised bed drying, drying temperature 50 DEG C.
4. the preparation method of composite bio-fertilizer according to claim 3, is characterized in that, fermentation of bacillus subtilis liquid adopts slant strains to spread cultivation step by step acquisition, and step is as follows:
(1) first order seed is cultivated: subtilis slant strains accessed in 500 ml shake flasks, substratum loading amount 100 milliliters, rotary shaker 180 revs/min, culture temperature 30 DEG C, incubation time 24 hours;
(2) secondary seed cultivate: by first order seed according to 10% inoculum size access in 500 milliliters of secondary seed shaking flasks, 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 flask with 10% inoculum size, substratum loading amount 1000 milliliters, rotary shaker 100 revs/min, culture temperature 30 DEG C, incubation time 24 hours;
(4) first class seed pot is cultivated: the first class seed pot being 150L with 10% inoculum size access cubic capacity by three grades of seeds, fermention medium loading amount 100L, culture temperature 28 DEG C, stirring velocity 100 revs/min, 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 is accessed cubic capacity with 10% inoculum size, fermention medium loading amount 1 ton, culture condition culture temperature 28 DEG C, stirring velocity 100 revs/min, ventilation (V/V) 1:0.5, tank pressure 0.05MPa, incubation time 24 hours.
5. the preparation method of composite bio-fertilizer according to claim 4, it is characterized in that, bacillus subtilis bacterium culture medium consists of: glucose 6%, yeast extract 1%, peptone 0.2%, CaCO
31%, pH6.8.
6. the preparation method of composite bio-fertilizer according to claim 1, it is characterized in that, preparation method is as follows for aspergillus niger culture: spawn culture, adopt conventional solid fermentation culture method: spore liquid is inoculated in solid state fermentation culture material, 26-35 DEG C is cultured to mycelia and covers with culture material, low temperature fluidized bed dry, pulverize dry thing.
7. the preparation method of composite bio-fertilizer according to claim 6, it is characterized in that, preparation method is as follows for aspergillus niger culture:
Slant strains activation culture: be transferred on slant medium by aspergillus niger slant strains, cultivates 3 days for 27 DEG C;
Solid first order seed is cultivated: the access of picking aspergillus niger slant strains is equipped with in 500 milliliters of triangular flasks of 100 grams of substratum and is carried out seed culture, cultivates 3 days for 30 DEG C;
Solid secondary seed is cultivated: stirred by above-mentioned cultured solid first order seed and carry out seed culture, culture condition for adding in 5000 milliliters of triangular flasks that 1000 grams of substratum are housed after fragment: cultivate 3 days for 30 DEG C;
Solid fermentation is cultivated: pulverized by second-level shake flask seed, add in the fermentation vat or pallet that sterilising medium is housed and mix rear cultivation, and bent material culture temperature controls at 26-35 DEG C, humidity 80-90%, every 10 hours stirrings once, and incubation time 5-7 days; The cultivation of solid koji material adopts conventional bent material culture technique; Treat culture material cover with mycelia can terminate cultivate, substratum in advance through high temperature steaming sterilising treatment, sterilising conditions control temperature 121 DEG C, 1 hour time;
Drying and crushing: fermentation ends culture material carries out drying on fluidized-bed or other drying plants, drying temperature controls, at 60 DEG C, to be dried to moisture content below 10%, then to be pulverized by solid culture medium, and crushing material aperture is more than 60 orders.
8. the preparation method of composite bio-fertilizer according to claim 6 or 7, it is characterized in that, aspergillus niger substratum consists of: solid material adds equivalent tap water; Initial pH nature; Described solid material mass percent consists of: wheat bran 70%, the crop material 15% of pulverizing, soybean cake powder 5%, W-Gum 10%.
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| CN101914447A (en) * | 2010-07-31 | 2010-12-15 | 大连三科生物工程有限公司 | Complex microbial inoculum 707 and preparation method and application thereof |
| CN102212494A (en) * | 2011-04-12 | 2011-10-12 | 王颖 | Organic matter decomposing inoculant, and preparation method and application thereof |
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| CN101914447A (en) * | 2010-07-31 | 2010-12-15 | 大连三科生物工程有限公司 | Complex microbial inoculum 707 and preparation method and application thereof |
| CN102212494A (en) * | 2011-04-12 | 2011-10-12 | 王颖 | Organic matter decomposing inoculant, and preparation method and application thereof |
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