CN104805028A - Micro-ecological fertilizer - Google Patents
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- CN104805028A CN104805028A CN201410034894.6A CN201410034894A CN104805028A CN 104805028 A CN104805028 A CN 104805028A CN 201410034894 A CN201410034894 A CN 201410034894A CN 104805028 A CN104805028 A CN 104805028A
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Abstract
The present invention relates to a micro-ecological fertilizer, and belongs to the technical field of agricultural fertilizers, wherein the micro-ecological fertilizer comprises, by weight, a phosphate-solubilizing Bacillus megaterium agent, a Bacillus subtilis culture, a Aspergillus niger culture, and a Lactobacillus plantarum agent, wherein the preservation number of the Bacillus subtilis Li-2013-02 is CGMCC No.7926, the Bacillus subtilis culture preparation method comprises culturing Bacillus subtilis through slant rotating culture, carrying out gradual expansion culture, transferring and inoculating the obtained seed liquid into a fermentation tank, and carrying out plate-frame filtration and drying on the fermentation broth after completing the fermentation so as to obtain the culture containing the Bacillus subtilis agent and the enzyme preparation, and characteristics of soil physical property improving and easy soil fertility improving are provided.
Description
Technical field
The present invention relates to a kind of fertilizer, belong to agricultural fertilizer technical field.
Background technology
The efficacy exertion of microbial fertilizer is mainly by the synergism to traditional fertilizer, fertilizer, and to the improvement activation of soil, and the mode such as the physiological action of microorganism realizes.Microorganism fertilizer a kind ofly causes farm crop to obtain the living microorganisms goods of specific fertilizer effect with microbial life activity and product thereof, it at culture fertility, improve chemical fertilizer utilization ratio, suppress the absorption of the objectionable impurities such as farm crop heavy metal and agricultural chemicals, have irreplaceable effect in utilizations of becoming thoroughly decomposed, raising quality of agricultural product etc. of purification and rehabilitating soil, promotion agricultural crop straw and municipal wastes.
Whether beneficial microorganism kind in microbial fertilizer, vital movement vigorous is the basis of its validity, and unlike other fertilizer be by the form of the principal elements such as nitrogen, phosphorus, potassium and how many based on.Just because of microbial fertilizer is preparation of living, thus its fertilizer efficiency and number of viable, intensity and ambient environmental conditions closely related, comprise temperature, moisture, potential of hydrogen, nutritional condition and formerly live in indigenous microorganism repulsive interaction in soil and have certain influence.
Aspergillus niger, be distributed widely in grain all over the world, plant product and soil, its product is harmless to human-body safety, is important fermentation industry bacterial classification.Such as application number is that the patent of invention of " 201110385820.3 " discloses a kind of aspergillus niger and carries out solid state fermentation and prepare soluble corn peptide, solid state fermentation is adopted to cultivate without the need to concussion in culturing process, culture condition is simple compared to liquid state fermentation, greatly can improve the reaction efficiency of product, reduce costs.
Due to China for a long time in microbial fertilizer research lack and drop into, the microbial fertilizer industry of China is still existed, and integral level is not high, technological innovation is not enough, quality product and effect show understable problem.Today of human consensus has been become, " bottleneck " that these problems have become microorganism fertilizer industry letter to be got through at agricultural sustainable development.
Summary of the invention
Technical problem to be solved by this invention provides a kind of fertilizer:
The parts by weight of fertilizer consist of: phosphorus decomposing bacillus megaterium microbial inoculum 1-5 part, subtilis culture 16-25 part, aspergillus niger culture 10-18 part, plant lactobacillus agent 5-12 part.
Described subtilis culture preparation: cultivate subtilis from inclined-plane switching, seed liquor after spreading cultivation step by step is transferred in fermentor tank, and the complete fermented liquid that ferments obtains the culture containing bacillus subtilis microbial agent and zymin after Plate Filtration, drying.
The preparation method of plant lactobacillus agent: from inclined-plane switching culturing plants Bacterium lacticum, 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 45% 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: 1, and vehicle group becomes: CaCO
320-23 part, dextrin 10-12 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 26-33 DEG C is cultured to mycelia and covers with culture material, and low temperature fluidized bed dry, pulverize dry thing; Common solid state fermentation is adopted 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, and seed dressing or front earth's surface of pouring water vegetative period are sprayed.
Beneficial effect
Fertilizer nutrient of the present invention is enriched, and organic content is high.The need of production of Organic food can be met.
Fertilizer of the present invention is formed by compound microorganism ferments, can improve the micro-ecological environment of soil, for the growth 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 improve 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, soil aggregate can be improved, strengthen the physicals of soil and reduce the loss of soil particle, under certain conditions, soil ulmin can also be participated in and formed.So use microbial fertilizer can improve soil physical property, be conducive to increasing soil fertility.
Embodiment:
Unless stated otherwise, technique means used in the present invention 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 only limited by claims.To those skilled in the art, under the prerequisite not deviating from essence of the present invention and scope, the various change carry out the reaction conditions in these embodiments, separation and Extraction condition 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.
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.
The agent of phosphorus decomposing bacillus megaterium 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.
Rui Gu bio tech ltd, Baoding provides colloid bacillus cereus bacterium powder.
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 (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 CGMCC No.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 (Bacillus subtilis) Li-2013-02 is preserved in the product Thermostable α-Amylase in Tianjin University of Technology 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)
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%, ammonium sulfate 0.4%, dipotassium hydrogen phosphate 0.8%, calcium chloride 0.2%.
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 Aspergillus niger Li-2013-03 provided by the invention takes turns mutagenesis screening by the aspergillus niger Aspergillus niger Li-2010 of Tianjin University of Technology's Laboratories Accession-strain cellulase-producing through nitrosoguanidine more, then obtains through leavening property test screen the Aspergillus niger strain Aspergillus nigerLi-2013-03 producing high activity cellulase to strain excellent.
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, city of BeiJing, China institute, postcode 100101), preserving number is CGMCCN0.7927.
Aspergillus niger strain Aspergillus niger Li-2013-03 (CGMCC No.7927) of the present invention 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, and diameter 150-450 μm, conidiophore betides matrix.Born of the same parents obstruct stem 1000-3000 (length) × 12-20 (diameter) μm, yellow or tawny, and wall is level and smooth; Spherical or the almost spherical of top capsule, diameter 45-75 μm, surface can be educated comprehensively; Produce born of the same parents' structure double-deck, metulae 10-20 (length) × 4.5-7.0 (diameter) μm, bottle stalk 6-10 (length) × 2.5-3.5 (diameter) μm; conidium is spherical or subsphaeroidal; diameter 3-4.5 μm, brown, wall is coarse.
2, feature is cultivated:
Bacterial strain grows rapidly on wort agar substratum, and 28 DEG C 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 yellowish.
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 DEG C, the suitableeest product enzyme temperature range 28-30 DEG C.
The triage techniques route of Aspergillus niger strain CGMCC No.7927 of the present invention is: the preparation → mutagenic treatment → plate isolation → primary dcreening operation → multiple sieve → genetic stability of starting strain → slant culture → spore suspension measures → expand experiment (leavening property mensuration).
By 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, the circumscribed beta-glucanase of the cellulase after 96 hours of fermenting, Endo-β-glucanase, beta-glucosidase and filter paper enzyme activity reach 620U/mL, 1289U/mL, 456U/mL and 732U/mL respectively.
28 DEG C of fermentations, 4 days diameter 75mm, fermented liquid cellulase circumscribed beta-glucanase, 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 than starting strain respectively.
Produce the screening method of high activity cellulase bacterial strain, comprise the following steps:
1) slant culture: by original Aspergillus niger strain Aspergillus niger Li-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
0brix wort 1000mL, 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 sterilized water Shen, 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 spergillus niger Li-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 spergi/lus nigerLi-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.
Embodiment 1
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 accessed in 500 ml shake flasks, substratum loading amount 100 milliliters, rotary shaker 180 revs/min, culture temperature 35 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 35 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 35 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 35 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.
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 is accessed cubic capacity with 5% inoculum size, fermention medium loading amount 2 tons, culture condition culture temperature 28 DEG C, tank pressure 0.05Mpa, incubation time 22 hours.The complete fermentation liquor low-temperature negative-pressure that ferments is concentrated in vacuo to 45% 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: 1, and vehicle group becomes: CaCO
325 parts, 12 parts, dextrin.Fluidised bed drying, drying temperature 50 DEG C.
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%, K
2hPO
40.2%, MgSO
4.7H
2o0.02%, MnSO
4.H
200.005%, CaCO
32%, agar 1.5%, pH6.8.
Plant lactobacillus bacterial classification is CICC20261.
Embodiment 2
The parts by weight of fertilizer consist of: phosphorus decomposing Bacillus megaterium microbial inoculum 3 parts, subtilis culture 20, aspergillus niger culture 15, plant lactobacillus agent 10 parts.
Embodiment 3: the parts by weight of fertilizer consist of: phosphorus decomposing Bacillus megaterium microbial inoculum 1 part, subtilis culture 25, aspergillus niger culture 18, plant lactobacillus agent 5 parts.
Embodiment 4: the parts by weight of fertilizer consist of: phosphorus decomposing Bacillus megaterium microbial inoculum 5 parts, subtilis culture 25, aspergillus niger culture 10, plant lactobacillus agent 12 parts.
Preparation method: mix according to aforementioned proportion, if desired can granulation.
Product effect experimental
Selection experimental field and test design: test and carry out in Ba Bao village, dapple pond town, Yanchi county Ningxia 27 days-October 30 March in 2009.
Experimental plot reaches field maize planting 10 mu, using product every mu of the present invention 0.6 kilogram respectively, emerging about 1 month and using invention product 0.4 kilogram by loose ground mode when planting, and control group uses common fertilizer.
Invention product uses 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 reaches 420 kilograms, improves 20% than control group per unit area yield.And experimental plot Soil structure is good, without bulk with harden.
Claims (7)
1. a fertilizer, is characterized in that, the parts by weight of described fertilizer consist of: phosphorus decomposing bacillus megaterium microbial inoculum 1-5 part, subtilis culture 16-25 part, aspergillus niger culture 10-18 part, plant lactobacillus agent 5-12 part.Described subtilis deposit number CGMCC No.7926, described aspergillus niger preserving number CGMCC No.7927.
2. a kind of microorganism fertilizer according to claim 1, is characterized in that, the preparation method of described subtilis culture 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.
Substratum forms: glucose 6%, yeast extract 1%, peptone 0.2%, CaCO
31%, pH6.8.
3. fertilizer according to claim 1, it is characterized in that, described aspergillus niger culture preparation method is as follows.Bacterial classification adopts solid fermentation to cultivate: spore liquid is inoculated in solid state fermentation culture material, and 26-33 DEG C is cultured to mycelia and covers with substratum, and low temperature fluidized bed dry, pulverize dry thing; Common solid state fermentation is adopted to prepare.
4. fertilizer according to claim 1, it is characterized in that, the parts by weight of institute's fertilizer consist of: phosphorus decomposing Bacillus megaterium microbial inoculum 3 parts, subtilis culture 20 parts, aspergillus niger culture 15 parts, plant lactobacillus agent 10 parts.
5. fertilizer according to claim 1, it is characterized in that, the parts by weight of institute's fertilizer consist of: phosphorus decomposing Bacillus megaterium microbial inoculum 1 part, subtilis culture 25, aspergillus niger culture 18, plant lactobacillus agent 5 parts.
6. fertilizer according to claim 1, it is characterized in that, the parts by weight of institute's fertilizer consist of: phosphorus decomposing Bacillus megaterium microbial inoculum 5 parts, subtilis culture 25, aspergillus niger culture 10, plant lactobacillus agent 12 parts.
7. according to claim 1-6, high-effective microorganism is fertile, and it is characterized in that, the preparation method of affiliated fertilizer is as follows: proportionally mixed by various microbial inoculum, or granulates after mixing; The preparation method of described subtilis culture is as follows: 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;
Described aspergillus niger culture preparation method is as follows: spawn culture, and solid fermentation is cultivated: spore liquid is inoculated in solid state fermentation culture material, and 26-33 DEG C is cultured to mycelia and covers with culture material, and low temperature fluidized bed dry, pulverize dry thing; Common homomorphism fermentation method is adopted to prepare;
The preparation method of described 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 is accessed cubic capacity with 5% inoculum size, fermention medium loading amount 2 tons, culture condition culture temperature 28 DEG C, tank pressure 0.05Mpa, incubation time 22 hours.The complete fermentation liquor low-temperature negative-pressure that ferments is concentrated in vacuo to 45% 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: 1, and vehicle group becomes: CaCO
325 parts, 12 parts, dextrin; Fluidised bed drying, drying temperature 50 DEG C;
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%, dipotassium hydrogen phosphate 0.2%, magnesium sulfate heptahydrate 0.02%, manganese sulfate monohydrate 0.005%, calcium carbonate 2%, agar 1.5%, pH6.8;
Plant lactobacillus bacterial classification is CICC20261.
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| CN201410034894.6A CN104805028A (en) | 2014-01-23 | 2014-01-23 | Micro-ecological fertilizer |
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| CN201410034894.6A Pending CN104805028A (en) | 2014-01-23 | 2014-01-23 | Micro-ecological fertilizer |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106119161A (en) * | 2016-06-30 | 2016-11-16 | 无锡市华东电力设备有限公司 | Composite bio-fertilizer and production method thereof |
| CN108947673A (en) * | 2018-08-14 | 2018-12-07 | 侯希波 | A kind of soil improvement Natural Circulation microbial bacterial agent and preparation method thereof |
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| WO2012083346A1 (en) * | 2010-12-23 | 2012-06-28 | Ams Laboratories Pty Ltd | Inoculum and method of preparation |
| CN103304285A (en) * | 2013-06-21 | 2013-09-18 | 南京宁粮农业科技有限公司 | Microbial agent and preparation method as well as application thereof |
| CN103739319A (en) * | 2013-12-30 | 2014-04-23 | 邵素英 | Microbial fertilizer |
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| WO2012083346A1 (en) * | 2010-12-23 | 2012-06-28 | Ams Laboratories Pty Ltd | Inoculum and method of preparation |
| CN102212494A (en) * | 2011-04-12 | 2011-10-12 | 王颖 | Organic matter decomposing inoculant, and preparation method and application thereof |
| CN103304285A (en) * | 2013-06-21 | 2013-09-18 | 南京宁粮农业科技有限公司 | Microbial agent and preparation method as well as application thereof |
| CN103739319A (en) * | 2013-12-30 | 2014-04-23 | 邵素英 | Microbial fertilizer |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106119161A (en) * | 2016-06-30 | 2016-11-16 | 无锡市华东电力设备有限公司 | Composite bio-fertilizer and production method thereof |
| CN108947673A (en) * | 2018-08-14 | 2018-12-07 | 侯希波 | A kind of soil improvement Natural Circulation microbial bacterial agent and preparation method thereof |
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