CN103739320A - Novel micro-ecological fertilizer - Google Patents
Novel micro-ecological fertilizer Download PDFInfo
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- CN103739320A CN103739320A CN201310743518.XA CN201310743518A CN103739320A CN 103739320 A CN103739320 A CN 103739320A CN 201310743518 A CN201310743518 A CN 201310743518A CN 103739320 A CN103739320 A CN 103739320A
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- 239000003337 fertilizer Substances 0.000 title claims abstract description 38
- 241000228245 Aspergillus niger Species 0.000 claims abstract description 32
- 244000063299 Bacillus subtilis Species 0.000 claims abstract description 6
- 235000014469 Bacillus subtilis Nutrition 0.000 claims abstract description 6
- 241000186660 Lactobacillus Species 0.000 claims description 12
- 239000003795 chemical substances by application Substances 0.000 claims description 12
- 229940039696 lactobacillus Drugs 0.000 claims description 12
- 244000005700 microbiome Species 0.000 abstract description 11
- 239000002689 soil Substances 0.000 abstract description 11
- 238000004519 manufacturing process Methods 0.000 abstract description 5
- 230000012010 growth Effects 0.000 abstract description 3
- 241000607479 Yersinia pestis Species 0.000 abstract description 2
- 150000001875 compounds Chemical class 0.000 abstract description 2
- 201000010099 disease Diseases 0.000 abstract description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 2
- 235000015097 nutrients Nutrition 0.000 abstract description 2
- 235000013348 organic food Nutrition 0.000 abstract description 2
- 240000006024 Lactobacillus plantarum Species 0.000 abstract 1
- 235000013965 Lactobacillus plantarum Nutrition 0.000 abstract 1
- 208000027418 Wounds and injury Diseases 0.000 abstract 1
- 230000006378 damage Effects 0.000 abstract 1
- 208000014674 injury Diseases 0.000 abstract 1
- 229940072205 lactobacillus plantarum Drugs 0.000 abstract 1
- 239000005416 organic matter Substances 0.000 abstract 1
- 108090000790 Enzymes Proteins 0.000 description 23
- 102000004190 Enzymes Human genes 0.000 description 23
- 230000001580 bacterial effect Effects 0.000 description 23
- 229940088598 enzyme Drugs 0.000 description 23
- 241000894006 Bacteria Species 0.000 description 19
- 230000000694 effects Effects 0.000 description 18
- 239000007788 liquid Substances 0.000 description 16
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- 239000002609 medium Substances 0.000 description 11
- 238000000034 method Methods 0.000 description 11
- 238000002360 preparation method Methods 0.000 description 11
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- 239000000243 solution Substances 0.000 description 8
- 108010059892 Cellulase Proteins 0.000 description 7
- 229940106157 cellulase Drugs 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- 238000010790 dilution Methods 0.000 description 7
- 239000012895 dilution Substances 0.000 description 7
- 239000000725 suspension Substances 0.000 description 7
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- 229940024171 alpha-amylase Drugs 0.000 description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 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
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
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- 239000004375 Dextrin Substances 0.000 description 2
- 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 2
- 108010059820 Polygalacturonase Proteins 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
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- 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
- KIWBPDUYBMNFTB-UHFFFAOYSA-N Ethyl hydrogen sulfate Chemical compound CCOS(O)(=O)=O KIWBPDUYBMNFTB-UHFFFAOYSA-N 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 238000003794 Gram staining Methods 0.000 description 1
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- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
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- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
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Abstract
The invention discloses a novel micro-ecological fertilizer, and belongs to the technical field of agricultural fertilizers. The micro-ecological fertilizer consists of the following components in parts by weight: 20 to 28 parts of bacillus subtilis culture, 15 to 25 parts of aspergillus niger culture and 8 to 15 parts of lactobacillus plantarum. The fertilizer is nutrient-rich and high in organic matter content, and the production requirements of organic food can be met; the fertilizer is obtained by fermenting compound microorganisms, so that the micro-ecological environment of soil can be improved, good conditions for the growth of crops can be provided, and the disease and pest injury resistance of the crops can also be enhanced.
Description
Technical field
The present invention relates to a kind of fertilizer, belong to agricultural fertilizer technical field.
Background technology
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.
A kind of Aspergillus niger strain of Chinese patent and the application in solid-state fermentation of pectase is produced thereof; application number: 200410006199.5; this invention relates to a kind of Aspergillus niger strain and the application in solid state fermentation is produced thereof, and the deposit number of its Aspergillus niger strain at China Committee for Culture Collection of Microorganisms's common micro-organisms center is CGMCC.NO.1100; The solid state fermentation that microorganism strains of the present invention can be used for polygalacturonase is cultivated aspergillus niger F02 bacterial strain of the present invention (Aspergillus niger F02) nutrition source in producing has carbon source, nitrogenous source, inorganic salt and trace nutrient; Adopting bacterial strain raw materials for production of the present invention is mainly the agricultural byproducts such as wheat bran, rice bran and flour, therefore less investment, easy to operate; With unit volume, calculate, the vigor of solid state fermentation polygalacturonase than the high 5-6 of liquid state fermentation doubly.
Microorganism fertilizer body level is not high, technological innovation is not enough, quality product and effect show understable problem.At agricultural sustainable development, become today of human consensus, these problems have become microorganism fertilizer industry letter " bottleneck " to be got through.
Summary of the invention
Technical problem to be solved by this invention provides a kind of novel fertilizer:
The parts by weight of fertilizer consist of: subtilis culture 20-28, aspergillus niger culture 15-25, plant lactobacillus agent 8-15 part.
Described subtilis culture preparation: cultivate subtilis from inclined-plane switching, seed liquor after spreading cultivation is step by step transferred in fermentor tank, and the complete fermented liquid that ferments obtains through Plate Filtration, after dry the culture that contains bacillus subtilis microbial agent and zymin.
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
320-23 part, dextrin 10-12 part.Fluidised bed drying, 50 ℃ of drying temperatures.
Aspergillus niger culture preparation: spawn culture, solid fermentation is cultivated: spore liquid is inoculated in 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; Adopt common solid state fermentation to prepare.
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.
Beneficial effect
Fertilizer of the present invention is nutritious, and organic content is high.Can meet the need of production of Organic food.
Fertilizer of the present invention is formed by compound microorganism ferments, can improve the micro-ecological environment of soil, for the growths of farm crop provides beneficial conditions.
Fertilizer of the present invention can strengthen the diseases and insect pests resistance of crop.
Fertilizer of the present invention can be improved soil.In fertilizer, beneficial microorganism can produce glucide, account for 0.1% of the soil organism, with plant mucilage, mineral idiosome and organic colloid combine, 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 and form.So use microbial fertilizer, can improve soil physical property, be conducive to increase soil fertility.
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.
Subtilis provided by the invention (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 (be called for short CGMCC, address is: No. 3, No. 1, North Star West Road, Chaoyang District, BeiJing, China city institute), 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, 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 preserved in the high temperature resistant α-amylase of product in Tianjin University of Technology laboratory 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, is stable superior strain, and enzyme work reaches 30000U/mL, than original strain enzyme, lives and improves 1.6 times.
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%, NaCl1%.
Described fermention medium: Semen Maydis powder 5%～15%, soybean cake powder 4%～10%, (NH) 2SO40.4%, K2HPO40.8%, CaCl20.2%.
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.
By bacterial strain Li-2013-02 fermentation, obtained 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 ℃, and better 110 ℃ of following temperature stabilities of preserving, and 115 ℃ 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.
1, the present invention uses the method for UV-LiCl-ethyl sulfate complex mutation to obtain the subtilis Li-2013-02 of a strain high-yield thermostable α-amylase, and this bacterial strain has acidproof, thermotolerance by force, produces the high feature of enzyme activity.
2, there is the high temperature resistant α-amylase enzyme activity of this bacterial strain production gained up to 30000-35000u/ml; Applicable temperature scope is 25-115 ℃, 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, be 3.0 o'clock enzyme complete stabilities alive, optimal reaction pH value is 4.2, higher than existing high temperature resistant α-amylase enzyme activity, enzyme effect optimum pH wide scope, heatproof degree is high, is particularly suitable for that temperature of reaction is high, liquefaction process and Mashing process the industrialization demand of depositing.
The aspergillus niger Aspergillus niger Li-2010 of one strain cellulase-producing of aspergillus niger Aspergillus niger Li-2013-03 provided by the invention Shi You Tianjin University of Technology laboratory preservation takes turns mutagenesis screening through nitrosoguanidine more, then strain excellent is obtained producing the Aspergillus niger strain Aspergillus niger Li-2013-03 of high activity cellulase through leavening property test screen.
The bacterial strain of the high activity cellulase of product provided by the invention is specially aspergillus niger Aspergillus niger Li-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, BeiJing, China city institute, postcode 100101), preserving number is CGMCC NO.7927.
Aspergillus niger strain Aspergillus niger Li-2013-03 of the present invention (CGMCC No.7927) has following microbial characteristic:
1, morphological feature:
Aspergillus niger strain CGMCC No.7927, biology morphology is for comprising conidium, born of the same parents' stalk, top capsule, producing several parts such as born of the same parents' structure.Conidial head is spherical to Radiation, diameter 150-450 μ m, and conidiophore betides matrix.Born of the same parents obstruct stem 1000-3000 (length) * 12-20 (diameter) μ m, yellow or tawny, and wall is level and smooth; Spherical or the almost spherical of top capsule, diameter 45-75 μ m, surface can be educated comprehensively; Produce born of the same parents' structure double-deck, metulae 10-20 (length) * 4.5-7.0 (diameter) μ m, bottle stalk 6-10 (length) * 2.5-3.5 (diameter) μ m, conidium is spherical or subsphaeroidal, diameter 3-4.5 μ m, brown, wall is coarse.
2, cultivate and learn feature:
Bacterial strain is grown rapidly on wort agar substratum, and 28 ℃ of 4 days spores can be paved with inclined-plane; Quality velvet shape or be slightly with cotton-shaped; Conidium structure is a large amount of, and brown-black, without transudate; Bacterium colony reverse side is slightly yellow.
3, physiological and biochemical property:
Aspergillus niger strain CGMCC No.7927 can grow in the carbon sources such as maize straw, straw, wood chip, potato, Semen Maydis powder, Zulkovsky starch, molasses, optimal pH scope 5-6, optimum growth temperature scope 28-33 ℃, the suitableeest product enzyme temperature range 28-30 ℃.
The triage techniques route of Aspergillus niger strain CGMCC No.7927 of the present invention is: experiment (leavening property mensuration) is measured → expanded to the preparation → mutagenic treatment → plate isolation → primary dcreening operation of starting strain → slant culture → spore suspension → multiple sieve → genetic stability.
Press mutagenesis screening scheme, mutant strain step-sizing is eliminated, finally to strain excellent through leavening property test screen, obtain a plant height and produce enzyme performance bacterial strain black-koji mould Aspergillus niger Li-2013-03, circumscribed beta-glucanase, Endo-β-glucanase, beta-glucosidase and the filter paper enzyme activity of cellulase after 96 hours that ferment reaches respectively 620U/mL, 1289U/mL, 456U/mL and 732U/mL.
4 days diameter 75mm of 28 ℃ of fermentations, fermented liquid cellulase circumscribed beta-glucanase, Endo-β-glucanase, beta-glucosidase and filter paper enzyme activity reach respectively 620U/mL, 1289U/mL, 456U/mL and 732U/mL, than starting strain, improved 9.21 times, 7.43 times, 8.15 times and 10.31 times respectively.
The screening method that produces high activity cellulase bacterial strain, comprises the following steps:
1) slant culture: by original Aspergillus niger strain Aspergillus niger Li-2010 streak inoculation slant medium, cultivate 2～3d for 30 ℃, until mycelium ripe, produce a large amount of black spores.Described slant medium is composed as follows: 12OBrix wort l000mL, pH value nature, 121 ℃ of sterilizing 20min;
2) spore suspension preparation (following steps all operate under aseptic condition): add 15mL sterilized water to test tube slant, spore is scraped, with filter paper filtering, pour filtered solution 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 106-107/mL.
B. 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.
C. 200rpm oscillatory reaction 30min at 30 ℃, the centrifugal 10min of 5000rpm collects thalline, with stroke-physiological saline solution washing for several times, stopped reaction.
D. suitably dilution is adjusted to 103/mL by spore concentration, gets last dilution bacterium liquid 0.2mL, and dilution spread is on Mierocrystalline cellulose-Congo red plate screening substratum.200 of 30 ℃ of cultivation bacterial strains that after 2～3 days, 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 ℃ of sterilizing 20min).
E. sieve again: the 200 strain bacterium that obtain are inoculated in to slant medium with aseptic toothpick respectively, and 30 ℃ are cultured to spore and are paved with inclined-plane.Respectively spore is fermented to be inoculated under aseptic washing to be equipped with in the 250mL triangular flask that 50mL sieves substratum again, inoculum size 10%(v/v), 30 ℃, 100r/min are cultivated 96h, measure respectively the cellulase activity of each bacterial strain.(described sieve again substratum 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 ℃ of sterilizing 20min).Choose the bacterial strain that cellulose enzyme vigor is the highest and carry 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 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.
5) scale-up
(1) seed culture: by the highest strains A spergillus nigerLi-2013-03 access 500mL triangular flask of cellulose enzyme vigor, 100 milliliters of seed culture medium loading amounts, 30 ℃, 150rpm shaking table cultivation 72-96h.
(2) seed tank culture: by seed liquor with 10%(v/v) inoculum size access is equipped with in the 10L fermentor tank of 7.5L fermented liquid, controlling pH value constant is 6.0 ± 0.2,30 ± 0.1 ℃ of culture temperature, 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 ℃ of sterilizing 20min.
After fermentation ends, get fermented supernatant fluid (crude enzyme liquid) and carry out enzyme activity detection after measured, circumscribed beta-glucanase, Endo-β-glucanase, beta-glucosidase and the filter paper enzyme activity of strains A spergillus niger Li-2013-03 reach respectively 620U/mL, 1289U/mL, 456U/mL and 732U/mL, have improved 9.21 times, 7.43 times, 8.15 times and 10.31 times respectively than starting strain Aspergillus niger Li-2010.
Embodiment 1
A composite fertilizer, parts by weight consist of: the parts by weight of fertilizer consist of: subtilis culture 24, aspergillus niger culture 18,12 parts of plant lactobacillus agent.
The preparation method of subtilis culture:
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 is accessed in 500 ml shake flasks to 100 milliliters of substratum loading amounts, 180 revs/min of rotary shaking tables, 35 ℃ 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, 35 ℃ 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, 35 ℃ 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, 35 ℃ 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.
Substratum forms: glucose 6%, yeast extract 1%, peptone 0.2%, CaCO31%, pH6.8.
The preparation method of plant lactobacillus agent:
Conventional seed fermentation cultural method obtains seed liquor;
Fermentor cultivation: it is 3 tons of tanks that first class seed pot bacterial classification be take to 5% inoculum size access cubic capacity, 2 tons of fermention medium loading amounts, 28 ℃ 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: CaCO325 part, 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%, Tween800.1%, K2HPO40.2%, MgSO4.7H2O0.02%, MnSO4.H2O0.005%, CaCO32%, agar 1.5%, pH6.8.
Plant lactobacillus bacterial classification is CICC20390.
The parts by weight of embodiment 2 fertilizers consist of: subtilis culture 20, aspergillus niger culture 25,15 parts of plant lactobacillus agent.
Embodiment 3: the parts by weight of fertilizer consist of: subtilis culture 25, aspergillus niger culture 20,10 parts of plant lactobacillus agent.
Embodiment 4: the parts by weight of fertilizer consist of: subtilis culture 20, aspergillus niger culture 25,15 parts of plant lactobacillus agent.
Preparation method: mix or granulation according to aforementioned proportion.
The experiment of product effect
Selection experimental field and test design: tested 27 days-October 30 March 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 0.6 kilogram every mu of product of the present invention respectively when plantation, emerges and by loose ground mode, uses 0.4 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 510 kilograms; This plot was at the 2nd year plant spring wheat, and spring wheat production has reached 420 kilograms, than control group per unit area yield, has improved 20%.And experimental plot Soil structure is good, without bulk and hardening.
Claims (5)
1. a novel fertilizer, the parts by weight of fertilizer consist of: subtilis culture 20-28, aspergillus niger culture 15-25, plant lactobacillus agent 8-15 part, subtilis (Bacillus subtilis) Li-2013-02 preserving number is CGMCC No.7926.
2. novel fertilizer according to claim 1, the parts by weight of fertilizer consist of: subtilis culture 20, aspergillus niger culture 25,15 parts of plant lactobacillus agent.
3. novel fertilizer according to claim 1, the parts by weight of fertilizer consist of: subtilis culture 25, aspergillus niger culture 20,10 parts of plant lactobacillus agent.
4. novel fertilizer according to claim 1, the parts by weight of fertilizer consist of: subtilis culture 20, aspergillus niger culture 25,15 parts of plant lactobacillus agent.
5. novel fertilizer according to claim 1, described aspergillus niger (Aspergillus niger) Li-2013-03 preserving number is CGMCC NO.7927.
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| CN104355689A (en) * | 2014-09-30 | 2015-02-18 | 天津天绿健科技有限公司 | Micro-ecological fertilizer and preparing method thereof |
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