CN103773719A - Preparation method of microbial compound fertilizer - Google Patents
Preparation method of microbial compound fertilizer Download PDFInfo
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- CN103773719A CN103773719A CN201310743756.0A CN201310743756A CN103773719A CN 103773719 A CN103773719 A CN 103773719A CN 201310743756 A CN201310743756 A CN 201310743756A CN 103773719 A CN103773719 A CN 103773719A
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- 238000002360 preparation method Methods 0.000 title claims abstract description 32
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a preparation method of a microbial compound fertilizer, and belongs to the technical field of an agricultural fertilizer. The preparation method of the microbial compound fertilizer is as follows: a colloid bacillus bacteria agent, a phosphate-solubilizing bacillus megaterium, a bacillus subtilis, an aspergillus niger culture and a lactobacillus plantarum are combined into the microbial compound fertilizer according to the ratio. The fertilizer disclosed by the invention is prepared by a compound microorganism fermentation method, the micro-ecological environment of the soil can be improved, and a beneficial condition is provided for growth of crops. Growth-promoting substances such as an auxin material and a gibberellin material can be secreted by the bacillus mucilaginosus in a composite strain, so as to increase roots and strengthen seedlings; a silicon element in the soil can be decomposed for utilization by a plant, so that a wax coat of the plant is thickened, and the water retaining capacity of the plant is improved. The aspergillus niger used in the preparation method has the characteristics of producing high-activity cellulose, and the cellulose is beneficial to decomposition of soil organic matters, formation of humus and release of carbon nutrition.
Description
Technical field
The present invention relates to a kind of bio-fertilizer working method, belong to agricultural fertilizer technical field, particularly a kind of micro-ecological composite fertilizer and preparation method thereof.
Background technology
Microorganism fertilizer is a kind ofly to cause farm crop to obtain the microorganism live body goods of specific fertilizer effect with microbial life activity and product thereof, it at culture fertility, improve chemical fertilizer utilization ratio, there is irreplaceable effect the aspect such as utilizations of becoming thoroughly decomposed, raising quality of agricultural product that suppresses absorption, purification and rehabilitating soil, promotion agricultural crop straw and the municipal wastes of farm crop to the objectionable impurities such as heavy metal and agricultural chemicals.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.
Therefore, microbial fertilizer has just had multiple fertility and effect of chemical fertilizer, fertilizer, beneficial organism bacterium, is activated fertilizer nutrient, improves effect of fertilizer, Crop Improvement quality, promotes the desirable fertilizer of agricultural produce synergy.Microbial fertilizer is live body fertilizer, and a large amount of beneficial microorganism vital movements that its effect mainly contains by it complete.Only have these beneficial microorganism vital movements of working as to complete.Only have when these beneficial microorganisms are in vigorous breeding and metabolic situation, Substance Transformation and useful meta-bolites could constantly form.Therefore, beneficial microorganism kind in microbial fertilizer, whether vital movement is vigorous is the basis of its validity, and is 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.
And agricultural microorganism microbial inoculum is in the market mainly single microbial strains, be difficult to reduce micro-ecologic structure, simultaneously a little less than the synergy of common bacterial classification and farm crop, unfavorable as improved stress resistance of plant and root system nutrition absorption, the patent application that for example publication No. is CN103374528A, disclose a kind of adopt prepared by aspergillus niger can efficient phosphate-solubilizing microbiobacterial agent, utilize aspergillus niger to there is stronger conversion capability to insoluble phosphate, and phosphorous organic compound (as plant element etc.) can be resolved into the characteristic of the titanium pigment that can be absorbed and used by plants.The patent application that for example publication No. is CN102888356A again, discloses a kind of method and application thereof that utilizes subtilis to prepare microbial fertilizer.Although there are in the market some complex micro organism fungicides, but itself or function singleness, or its using method is restricted, crop yield effect after use can't reach satisfactory, so still need to develop the complex micro organism fungicide or the fertilizer that are applicable to multiple route of administration, have comprehensive function and good effect of increasing production at present.
Because China lacks and drops in research aspect microbial fertilizer for a long time, make that the microbial fertilizer industry of China still exists that integral level is not high, technological innovation is not enough, quality product and effect show understable problem.Become today of human consensus at agricultural sustainable development, 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 is to overcome the deficiencies in the prior art, and a kind of preparation method of micro-ecological composite fertilizer is provided.
Technical solution of the present invention is: a kind of preparation method of micro-ecological composite fertilizer, is combined as micro-ecological composite fertilizer in proportion by colloid bacillus cereus microbial inoculum, phosphorus decomposing bacillus megaterium, subtilis, aspergillus niger culture and plant lactobacillus; Described micro-ecological composite fertilizer parts by weight consist of: colloid bacillus cereus microbial inoculum 3-6 part, phosphorus decomposing bacillus megaterium microbial inoculum 2-6 part, subtilis culture 10-15, aspergillus niger culture 8-15, plant lactobacillus agent 1-4 part.
Described subtilis culture preparation: cultivate subtilis from inclined-plane switching, the seed liquor after spreading cultivation is step by step transferred in fermentor tank, the complete fermented liquid that ferments obtains the culture containing bacillus subtilis microbial agent through Plate Filtration, after dry.
The preparation method of plant lactobacillus agent: from inclined-plane switching culturing plants Bacterium lacticum, the seed liquor after spreading cultivation is step by step transferred in fermentor tank, and the complete fermented liquid that ferments is concentrated in vacuo to 45% of original volume through low-temperature negative-pressure, obtains bacterium concentrated solution.Add carrier: in concentrated solution, add the carrier mixing, mix; The weight ratio of concentrated solution and carrier is 0.5-0.6:1, and vehicle group becomes: CaCO320-30 part, dextrin 10-15 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.
The bacterial classification that the present invention adopts is as follows:
Subtilis (Bacillus subtilis subsp) CGMCC7926
Plant lactobacillus (Lactobacillus plantarum) CGMCC7928
Aspergillus niger (Aspergillus niger) CGMCC NO.7927
The depositary institution of above-mentioned three kinds of bacterial classifications is Chinese common micro-organisms culture presevation administrative centers.Address: No. 3, No. 1, North Star West Road, Chaoyang District, city of BeiJing, China institute; Postcode: 100101.
Phosphorus decomposing bacillus megaterium is a kind in Bacillus (Bacillus).Motion, forms pod membrane, aerobic.Produce acid from glucose, also often produce acid from pectinose and N.F,USP MANNITOL.Hydrolyzed starch, does not produce lecithinase, VP feminine gender.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, international business affairs center 1 district 807-812.
Rui Gu bio tech ltd, Baoding provides colloid bacillus cereus bacterium powder.
Beneficial effect
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.
Colloid bacillus cereus claims again silicate bacteria, its key property is potassium, the silicon that can decomposite in the mineral such as feldspar, mica, also can decomposite the phosphorus in phosphatic rock, and secretion plant growth substance and plurality of enzymes, to strengthen the resistibility of crop to some diseases.Endobacillary potassium, after thalline death, dissociates out, can be absorbed and used by plants again.As a kind of critical function bacterium in microbial fertilizer, can improve Soil Available potassium, phosphorus content, improve the multiple effects such as crop yield and quality.
Colloid bud pole bacterium in composite bacteria of the present invention can secrete the growth-promoting substance such as growth hormone material and Plant hormones regulators,gibberellins material, extraneous root strong sprout; Decompose element silicon in soil and, for plant utilization, the wax layer of plant is thickened, improve plant water keeping ability; Can promote soil aggregate to form, keep soil from packing together; Spoiled soil capillarity, prevents soil water evaporation.
Industrial at bio-feritlizer, aspergillus niger has cracking larger molecular organics and indissoluble inorganics, is convenient to Crop utilization, improves Soil structure, strengthens soil fertility, improves the effect of crop yield.Aspergillus niger has the characteristic of phosphorus decomposing, can utilize the phosphorus of stationary state, ADSORPTION STATE, effectively alleviates the present situation that lacks phosphorus in soil, and to improving phosphate fertilizer utilization efficiency, the environmental pollution that the use of minimizing phosphate fertilizer causes.
The aspergillus niger that the present invention uses also has the characteristic of producing high activity cellulase, and cellulase is conducive to the decomposition of the soil organism, and the formation of soil ulmin and the release of carbon nutrition, be used on orchard crop, can make pulp delicacy clear and melodious, and fruit juice is abundant, good mouthfeel.
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 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, and can improve soil aggregate, strengthen the physicals of soil and the loss of minimizing soil particle, under certain conditions, can also participate in soil ulmin forms.
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.
Embodiment
Unless stated otherwise, in the present invention, technique means used is method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, but not limits the scope of the invention, and the spirit and scope of the invention are only limited by claims.To those skilled in the art, do not deviating under the prerequisite of essence of the present invention and scope, the various changes that the reaction conditions in these embodiments, separation and Extraction condition are carried out or change also belong to protection scope of the present invention.
The following examples can make the present invention of those skilled in the art comprehend, but do not limit the present invention in any way.
Embodiment 1
Subtilis (Bacillus subtilis) Li-2013-02, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on July 15th, 2013 and (is called for short CGMCC, address is: No. 3, No. 1, North Star West Road, Chaoyang District, city of BeiJing, China institute), preserving number is CGMCC No.7926.
Described strain characteristic is that the enzyme activity of the high temperature resistant α-amylase of product is high, heat-resisting, acid resistance is strong.
High temperature resistant α-amylase enzyme activity prepared by described bacterial strain is 30000-35000u/ml; Applicable temperature scope is 105-115 ℃, and 110 ℃ of optimal reactive temperatures, at 110 ℃ of enzymes complete stability alive; Being suitable for pH value in reaction scope is 3.0-7.0, is 3.0 o'clock enzyme complete stabilities alive in pH value, and optimal reaction pH value is 4.2.
Described bacterial strain feature is as follows:
Described bacterial strain colony colour on solid plate is oyster white, and surface drying is opaque, and neat in edge, for having the aerobic bacteria of mobility.Microscopy is elongated rod shape, and gramstaining is positive.This bacterium can utilize Citrate trianion, and nitrate reductase, V-P test into positive.
Described subtilis (Bacillus subtilis) Li-2013-02 is produced high temperature resistant α-amylase subtilis Li-2013 by a strain obtains through UV-LiCl-ethyl sulfate Mutation screening, specifically screens step as follows:
(1) preparation of bacteria suspension
By in mono-the Li-2013 growing after plate streaking separates bacterium colony 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, wrap with cloth or the newspaper of black, put 40 ℃ and cultivate 48h, on the flat board that grows bacterium colony, filtering out hydrolysis circle chooses to inclined-plane and preserves with colony diameter ratio the maximum, 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, on the flat board that grows bacterium colony, primary dcreening operation goes out hydrolysis circle and chooses to inclined-plane and preserve 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 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, is 1 enzyme activity unit, represents with U/mL.
After measured, bacterial strain Li-2013-02, be stable superior strain, and enzyme work reaches 30000U/mL.
Described lithium chloride flat board: 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%, (NH)
2sO
40.4%, K
2hPO
40.8%, CaCl
20.2%.
Described shake-flask culture condition: this bacterium in the 250mL shaking flask that contains 30mL fermention medium, inoculum size 10% (V/V), 100r/min, 40 ℃ of fermentation culture 72h.
Described high-temperature resistant alpha-amylase, its zymologic property is as follows:
(1) this enzyme thermal adaptation a wider range, optimum temperature is between 100-110 ℃, and 110 ℃ of following preserve, temperature stability is better, and it is poor more than 110 ℃ to preserve long-time temperature stability.
(2) this enzyme optimal reaction pH value is 4.2.Between pH value 3.0-7.0, all having high enzyme vigor, is 3.0 o'clock enzyme complete stabilities alive in pH value.
(3) enzymic activity: by mutant strain Li-2013-02 provided by the present invention, the high temperature resistant α-amylase enzyme activity of preparation is 30000-35000U/ml.
Plant lactobacillus provided by the present invention (Lactobacillus plantarum) Li-2013-01, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC No.7928, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101.Preservation date on July 15th, 2013.This bacterial strain feature is as follows: examine under a microscope, this bacterial strain is rod-short, and gramstaining is positive, and boundless hair, does not produce gemma; On solid medium, this bacterium bacterium colony is white, smooth surface, and densification, form is circular, edge is more neat.Physicochemical characteristics is: catalase (-), and gelatine liquefication (-), indoles experiment (+), mobility (-), fermentation gas (-), nitrite reduction (-), fermentation gas (-), produces hydrogen sulfide (-), growth (+) in pH4.5MRS substratum.
Plant lactobacillus of the present invention adopts following flow process to carry out seed selection:
Original sieve again → mitotic stability of bacterial classification → test tube activation → ethyl sulfate (DES) mutagenesis → dull and stereotyped primary dcreening operation → nitrosoguanidine (NTG) mutagenesis → dull and stereotyped primary dcreening operation → shaking flask test of setting out.
The original bacterial classification that sets out is CICC20242, is purchased from Chinese industrial microbial strains preservation administrative center.
Original strain of the present invention is in xylan substratum, and the output of lactic acid is 12.5g/L.In order to improve its lactic acid production, adopt successively DES and NTG to carry out mutagenesis to this bacterial classification, mutagenesis adopts MRS calcium carbonate flat board to carry out primary dcreening operation, then adopt 500mL shake flask fermentation, biosensor analysis instrument carries out multiple sieve to Producing Strain, the lactobacterium plantarum strain that seed selection is good, the experiment of then going down to posterity, evaluates its genetic stability.
Bacterial strain CGMCC No.7928 genetic stability result shows: through continuous passage ten times, property indices is all more stable, and heredity is better, and proterties is not replied, the object bacterial strain therefore bacterial strain CGMCC No.7928 being obtained as seed selection.
Object bacterial strain CGMCC No.7928 is done to the experiment of 10L fermentor tank, and result shows: after fermentation 72h, take xylan as carbon source, the lactic acid concn of plant lactobacillus CGMCC No.7928 can reach 57g/L, has improved 356% compared with starting strain.
Object bacterial strain CGMCC No.7928 is done to the experiment of 10L fermentor tank, and result shows: after fermentation 72h, take glucose as carbon source, the lactic acid concn of plant lactobacillus CGMCC No.7928 can reach 68g/L.
Detailed process is as follows:
Substratum:
Liquid MRS xylan substratum (extractum carnis 2g, peptone 10g, yeast extract paste 5g, xylan 20g, sodium acetate 5g, ammonium citrate 2g, dipotassium hydrogen phosphate 2g, magnesium sulfate heptahydrate 0.2g, seven water manganous sulfate 0.05g, after dissolving one by one, tap water constant volume 1000mL, regulates pH7.0-7.2); MRS xylan screening solid medium (extractum carnis 2g, peptone 10g, yeast extract paste 5g, xylan 90g, sodium acetate 5g, ammonium citrate 2g, dipotassium hydrogen phosphate 2g, magnesium sulfate heptahydrate 0.2g, seven water manganous sulfate 0.05g, after dissolving one by one, tap water constant volume 1000mL, regulate pH7.0-7.2, add 20g agar).
1. ethyl sulfate (DES) mutagenic and breeding
1) on super clean bench, get plant lactobacillus one ring on test tube slant, access is equipped with in the 250mL triangular flask of 50mL liquid MRS xylan substratum, and 200rpm cultivates 12h left and right for 40 ℃, makes thalline in logarithmic growth in earlier stage.
2) get 5mL bacterium liquid, the centrifugal 10min of 5000rpm collects thalline, with physiological saline washing 2 times.
3) be diluted to 107/mL bacteria suspension with pH7.0 phosphoric acid buffer.
4) potassium phosphate buffer, 8mL bacteria suspension, 0.4mLDES of getting 32mLpH7.0 fully mixes at the 150mL triangular flask of putting in advance rotor, and making DES ultimate density is 1%(v/v).
5) 150rpm reaction 30min in 30 ℃ of shaking tables, gets 1mL mixed solution, adds 0.5mL25%Na
2s
2o
3solution stopped reaction.
6) dilution spread is in the MRS xylan screening solid medium plate containing 90g/L xylan.At the bacterial strain of 40 ℃ of cultivations picking transparent circle/colony diameter maximum after 2～3 days, label is DES bacterium.
2. nitrosoguanidine mutagenesis
1) on super clean bench, get plant lactobacillus DES mono-ring on test tube slant, access is equipped with in the 250mL triangular flask of 50mL liquid MRS xylan substratum, and 200rpm cultivates 12h left and right for 40 ℃, makes thalline in logarithmic growth in earlier stage.
2) get the centrifugal 10min of 5mL bacterium liquid 5000rpm and collect thalline, with physiological saline washing 2 times.
3) be diluted to 107/mL bacteria suspension with pH6.0 phosphoric acid buffer.
4) get 10mL bacteria suspension and be transferred in 100mL triangular flask, add the NTG of 10mg, being mixed with final concentration is the NTG solution of 10mg/mL, and adds 4-5 to drip acetone, is beneficial to NTG and dissolves.
5) 200rpm oscillatory reaction 30min at 30 ℃, the centrifugal 10min of 5000rpm collects thalline, by stroke-physiological saline solution washing several, stopped reaction.
6) suitably dilution, gets last dilution bacterium liquid 0.2mL, and dilution spread is in the MRS xylan screening solid medium plate containing 90g/L xylan.150 of 40 ℃ of cultivation bacterial strains that after 2～3 days, picking transparent circle/colony diameter is larger.
3. shaking flask is sieved again
1) on super clean bench, get respectively plant lactobacillus one ring on each test tube slant, access is equipped with in the 250mL triangular flask of 50mL liquid MRS xylan substratum, and 200rpm cultivates 3-4 days, detects glucose concn and Pfansteihl change in concentration every day for 40 ℃.After fermentation ends, relatively the xylan wear rate of 150 strain bacterial classifications and lactic acid produce transformation efficiency and the heteroacid content of speed, lactic acid.
2) selection xylan metabolic rate is fast, lactic acid concn is high, transformation efficiency is high and the poor bacterial classification of heteroacid is final bacterial classification, called after Li bacterium.
4. genetic stability test
Li-2013-01 bacterial strain is gone down to posterity for continuous ten times on inclined-plane, and the method for sieving again by shaking flask detects the fermentation situation after at every turn going down to posterity.Experiment discovery is gone down to posterity for continuous ten times on inclined-plane, and this bacterial classification proterties does not have considerable change, and property indices is all normal, illustrates that the genetic stability of this bacterial classification is stronger.
Aspergillus niger Aspergillus niger Li-2013-03 provided by the invention takes turns mutagenesis screening by the aspergillus niger Aspergillus niger Li-2010 of a strain cellulase-producing of Tianjin University of Technology's laboratory preservation 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, city of BeiJing, China 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 bilayer, 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 → multiple sieve → genetic stability of starting strain → slant culture → spore suspension.
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, improved 9.21 times, 7.43 times, 8.15 times and 10.31 times than starting strain 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 maturation, 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
1. with sterilized water, spore suspension is adjusted to and is diluted to 106-107/mL.
2. 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.
3. 200rpm oscillatory reaction 30min at 30 ℃, the centrifugal 10min of 5000rpm collects thalline, by stroke-physiological saline solution washing several, stopped reaction.
4. suitably spore concentration is adjusted to 103/mL by dilution, 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).
5. 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 strains A spergillus niger Li-2013-03 access 500mL triangular flask the highest 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 2
A kind of micro-ecological composite fertilizer, parts by weight consist of: 5 parts of colloid bacillus cereus microbial inoculums, 3 parts of phosphorus decomposing bacillus megaterium microbial inoculums, 10 parts of subtilis cultures, 12 parts of aspergillus niger cultures, 2 parts of plant lactobacillus agent.
The preparation method of subtilis culture:
The acquisition of fermented liquid: adopt slant strains to spread cultivation step by step and obtain fermentation of bacillus subtilis liquid;
(1) first order seed is cultivated: subtilis slant strains is accessed in 500 ml shake flasks to 100 milliliters of substratum loading amounts, 180 revs/min of rotary shaking tables, 30 ℃ of culture temperature, incubation time 24 hours;
(2) secondary seed is cultivated: by first order seed, according in 500 milliliters of secondary seed shaking flasks of inoculum size access of 10%, culture condition is identical with first order seed;
(3) three grades of seed culture: secondary seed is accessed in 5000 milliliters of three grades of seed shaking flasks to 1000 milliliters of substratum loading amounts, 100 revs/min of rotary shaking tables, 30 ℃ of culture temperature, incubation time 24 hours with 10% inoculum size;
(4) first class seed pot is cultivated: the first class seed pot by three grades of seeds take 10% inoculum size access cubic capacity as 150L, fermention medium loading amount 100L, 28 ℃ of culture temperature, 100 revs/min of stirring velocitys, ventilation (V/V) 1:0.5, tank pressure 0.05MPa, incubation time 24 hours;
(5) fermentation culture: first class seed pot bacterial classification is accessed to cubic capacity as 1.5 tons of secondary seed tanks take 10% inoculum size, 1 ton of fermention medium loading amount, 28 ℃ of culture condition culture temperature, 100 revs/min of stirring velocitys, ventilation (V/V) 1:0.5, tank pressure 0.05MPa, incubation time 24 hours.Dry acquisition after fermented liquid Plate Filtration.
Substratum composition: glucose 6%, yeast extract 1%, peptone 0.2%, CaCO
31%, pH6.8.
The preparation method of plant lactobacillus agent:
(1) first order seed is cultivated: plant lactobacillus bacterial classification is accessed in 500 ml shake flasks to 100 milliliters of substratum loading amounts, 30 ℃ of culture temperature, incubation time 24 hours;
(2) secondary seed is cultivated: by first order seed, according in 500 milliliters of secondary seed shaking flasks of inoculum size access of 10%, culture condition is identical with first order seed;
(3) three grades of seed culture: secondary seed is accessed in 5000 milliliters of three grades of seed shaking flasks to 1000 milliliters of substratum loading amounts, 30 ℃ of culture temperature, incubation time 24 hours with 10% inoculum size;
(4) first class seed pot is cultivated: the first class seed pot by three grades of seeds take 5% inoculum size access cubic capacity as 150L, fermention medium loading amount 100L, 30 ℃ of culture temperature, tank pressure 0.05MPa, incubation time 18 hours;
(5) fermentor cultivation: first class seed pot bacterial classification is accessed to cubic capacity as 3 tons of secondary seed tanks take 5% inoculum size, 2 tons of fermention medium loading amounts, 30 ℃ of culture condition culture temperature, tank pressure 0.05MPa, incubation time 22 hours.The complete fermented liquid that ferments is concentrated in vacuo to 45% of original volume through low-temperature negative-pressure, obtains bacterium concentrated solution.Add carrier: in concentrated solution, add the carrier mixing, mix; The weight ratio of concentrated solution and carrier is 0.5:1, and vehicle group becomes: CaCO
325 parts, 12 parts, dextrin, fluidised bed drying, 50 ℃ of drying temperatures.
Substratum consists of: casein peptone 1%, beef extract 1%, yeast extract 0.5%, glucose 0.5%, sodium acetate 0.5%, citric acid diamines 0.2%, Tween800.1%, K
2hPO
40.2%, MgSO
4.7H
2o0.02%, MnSO
4.H
2o0.005%, CaCO
32%, agar 1.5%, pH6.8.
The preparation method of aspergillus niger culture:
Slant strains activation culture: aspergillus niger slant strains is transferred on slant medium, cultivates 3 days for 27 ℃;
Solid first order seed is cultivated: picking aspergillus niger slant strains accesses in 500 milliliters of triangular flasks that 100 grams of substratum are housed carries out seed culture, cultivates 3 days for 30 ℃;
Solid secondary seed is cultivated: above-mentioned cultured solid first order seed is stirred as adding after fragment and in 5000 milliliters of triangular flasks that 1000 grams of substratum are housed, carries out seed culture, culture condition: cultivate 3 days for 30 ℃;
Solid fermentation is cultivated: secondary shake-flask seed is pulverized, added to be equipped with in the fermentation vat of sterilising medium or pallet to mix rear cultivation, bent material culture temperature is controlled at 26-35 ℃, humidity 80-90%, every 10 hours stirrings once, incubation time 5-7 days; The cultivation of solid koji material adopts conventional bent material culture technique; Treat that culture material covers with mycelia and can finish to cultivate, substratum is in advance through high temperature steaming sterilising treatment, 121 ℃ of sterilising conditions control temperature, 1 hour time;
Drying and crushing: fermentation ends culture material is dried on fluidized-bed or other drying plants, and drying temperature is controlled at 60 ℃, is dried to moisture content below 10%, then solid culture medium is pulverized, and crushing material aperture is more than 60 orders;
Substratum composition: solid material: wheat bran 80%, soybean cake powder 10%, W-Gum 10%, adds equivalent tap water; Initial pH nature.
Embodiment 3
A kind of micro-ecological composite fertilizer, parts by weight consist of: 4 parts of colloid bacillus cereus microbial inoculums, 5 parts of phosphorus decomposing bacillus megaterium microbial inoculums, 14 parts of subtilis cultures, 13 parts of aspergillus niger cultures, 3 parts of plant lactobacillus agent.
Produce a method for micro-ecological composite fertilizer, colloid bacillus cereus microbial inoculum, phosphorus decomposing bacillus megaterium, subtilis, aspergillus niger culture and plant lactobacillus are combined as to micro-ecological composite fertilizer in proportion.
Bacillus subtilis microbial inoculum is cultivated preparation: cultivate subtilis from inclined-plane switching, the seed liquor after spreading cultivation is step by step transferred in fermentor tank, and the complete fermented liquid that ferments obtains subtilis culture through Plate Filtration, after dry.
The preparation 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 0.6:1, and vehicle group becomes: CaCO
330 parts, 16 parts, dextrin.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 30 ℃ are cultured to mycelia and cover with culture material, and 35 ℃ of low temperature fluidized-beds dry, pulverize dry thing.
Embodiment 4
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 0.5 kilogram every mu of product of the present invention respectively in the time of plantation, emerges and uses 0.5 kilogram of invention product by loose ground mode about 1 month, and control group uses conventional fertilizer.
Invention product uses corn field corn yield to reach 650 kilograms, and control group reaches 500 kilograms; The use of invention product makes corn per mu yield improve 30%.This plot was at the 2nd year plant spring wheat, and spring wheat production has reached 400 kilograms, has improved 20% than control group per unit area yield.And experimental plot Soil structure is good, without bulk and hardening.
Claims (7)
1. a preparation method for micro-ecological composite fertilizer, comprises the steps, colloid bacillus cereus microbial inoculum, phosphorus decomposing bacillus megaterium, subtilis, aspergillus niger culture and plant lactobacillus are combined as to micro-ecological composite fertilizer in proportion.
2. the preparation method of micro-ecological composite fertilizer according to claim 1, is characterized in that, parts by weight consist of: colloid bacillus cereus microbial inoculum 3-6 part, phosphorus decomposing bacillus megaterium microbial inoculum 2-6 part, subtilis culture 10-15, aspergillus niger culture 8-15, plant lactobacillus agent 1-4 part; Described subtilis preserving number is CGMCC No.7926, and described aspergillus niger deposit number is CGMCC NO.7927, and described plant lactobacillus deposit number is CGMCC No.7928.
3. the preparation method of micro-ecological composite fertilizer according to claim 1, it is characterized in that, the preparation method of described subtilis culture is as follows: 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 the culture containing bacillus subtilis microbial agent through Plate Filtration, after dry.
4. the preparation method of micro-ecological composite fertilizer according to claim 1, is characterized in that, the preparation method of described plant lactobacillus agent is as follows: the complete fermented liquid that ferments is concentrated in vacuo to 45% of original volume through low-temperature negative-pressure, obtains bacterium concentrated solution; Add carrier: in concentrated solution, add the carrier mixing, mix; The weight ratio of concentrated solution and carrier is 0.5-0.6:1, and vehicle group becomes: CaCO
320-30 part, dextrin 10-15 part, fluidised bed drying, 50 ℃ of drying temperatures.
5. the preparation method of micro-ecological composite fertilizer according to claim 1, it is characterized in that, the preparation method of described aspergillus niger culture is as follows: solid fermentation is cultivated end culture material and is dried on fluidized-bed or other drying plants, drying temperature is controlled at 60 ℃, be dried to moisture content below 10%, then solid culture medium is pulverized, crushing material aperture is more than 60 orders;
Substratum composition: solid material: wheat bran 80%, soybean cake powder 10%, W-Gum 10%, adds equivalent tap water; Initial pH nature.
6. a kind of preparation method of micro-ecological composite fertilizer according to claim 2, is characterized in that, parts by weight consist of: 5 parts of colloid bacillus cereus microbial inoculums, 3 parts of phosphorus decomposing bacillus megaterium microbial inoculums, 10 parts of subtilis cultures, 12 parts of aspergillus niger cultures, 2 parts of plant lactobacillus agent.
7. a kind of preparation method of micro-ecological composite fertilizer according to claim 2, is characterized in that, parts by weight consist of: 4 parts of colloid bacillus cereus microbial inoculums, 5 parts of phosphorus decomposing bacillus megaterium microbial inoculums, 14 parts of subtilis cultures, 13 parts of aspergillus niger cultures, 3 parts of plant lactobacillus agent.
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| CN104311169A (en) * | 2014-09-28 | 2015-01-28 | 邵素英 | Compound biological fertilizer and preparation method thereof |
| CN105198672A (en) * | 2015-09-28 | 2015-12-30 | 湖南泰谷生物科技股份有限公司 | Phosphorus- and potassium-solubilizing microorganism fertilizer and preparation method and application thereof |
| CN107011029A (en) * | 2017-04-06 | 2017-08-04 | 哈尔滨尼亚农业有限公司 | Seedling agent and its production method are strengthened in a kind of green acid adjustment of high-effective microorganism |
| CN111718876A (en) * | 2020-06-29 | 2020-09-29 | 北京中外建建筑设计有限公司 | Compound microbial agent and preparation method and application thereof |
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