CN103773720B - A kind of preparation method of microbial fertilizer - Google Patents
A kind of preparation method of microbial fertilizer Download PDFInfo
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- CN103773720B CN103773720B CN201310743759.4A CN201310743759A CN103773720B CN 103773720 B CN103773720 B CN 103773720B CN 201310743759 A CN201310743759 A CN 201310743759A CN 103773720 B CN103773720 B CN 103773720B
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- 238000002360 preparation method Methods 0.000 title claims abstract description 28
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- 241000186660 Lactobacillus Species 0.000 claims abstract description 27
- 229940039696 lactobacillus Drugs 0.000 claims abstract description 27
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims abstract description 21
- 229910052698 phosphorus Inorganic materials 0.000 claims abstract description 21
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- 238000000034 method Methods 0.000 claims abstract description 19
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Fertilizers (AREA)
Abstract
The invention discloses a kind of preparation method of microbial fertilizer, belong to agricultural fertilizer technical field, the production method of described microbial fertilizer is mixed in proportion aspergillus niger culture, phosphorus decomposing bacillus megaterium, plant lactobacillus and subtilis into microbial fertilizer.Fertilizer of the present invention is made by the method for compound microorganism ferments, can improve the micro-ecological environment of soil, for the growth of farm crop provides beneficial conditions.The phosphorus decomposing bacillus megaterium organophosphorus that can decompose in soil becomes the available rapid available phosphorus of plant; The aspergillus niger that the present invention uses has the characteristic of producing high activity cellulase, and cellulase is conducive to the decomposition of the soil organism, the formation of soil ulmin and the release of carbon nutrition.The amount of every mu of land use fertilizer of the present invention is 50-100 gram, is one of microbial fertilizer that usage quantity is less both at home and abroad at present.<pb pnum="1" />
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 preparation method of microbial fertilizer.
Background technology
Existing bio-feritlizer is monistic bio-feritlizer mostly.As: azotobacteria fertilizer, primarily of bacterium and the fungi composition with nitrogen fixation; And for example: phosphorus bacteria, its function decomposes the organophosphorus in soil, makes it to become the phosphorus element being easy to be absorbed by plants; For another example: B. mucilaginocus, its function decomposes the potassium compound in soil, makes it to become the available potassium that can be absorbed and used by plants.These fertilizer play each self-applying being used alone Shi Junneng, and part meets plant to nitrogen, phosphorus, the needs of potassium three elements.They can not produce the soil compaction phenomenon that life-time service chemical fertilizer causes.But its deficiency is: be used alone the aspect that above-mentioned three class bio-feritlizers can only solve plant desired nutritional, and cost is higher, consumption is large, meanwhile, by three with the use of, suitably cause again deficiency or the waste of rate of fertilizer application because being difficult to the ratio of accomplishing.Facts have proved of the bio-feritlizer of the single or compound of life-time service, they can only replacing fertilizer less than 30%.
And agricultural microbial agent is in the market mainly single microbial strains, be difficult to reduction Tiny ecosystem structure, the synergy of common bacterial classification and farm crop is weak simultaneously, unfavorable as improved stress resistance of plant and root system nutrition absorption, such as publication No. is the patent application of CN103374528A, disclose a kind of adopt aspergillus niger to prepare can the microbiobacterial agent of efficient phosphate-solubilizing, make use of aspergillus niger, to insoluble phosphate, there is stronger conversion capability, and phosphorous organic compound (as planting element etc.) can be resolved into the characteristic of the titanium pigment that can be absorbed and used by plants.Such as publication No. is the patent application of CN102888356A again, disclose a kind of utilize subtilis to prepare microbial fertilizer method and application.Although there are some complex micro organism fungicides in the market, but itself or function singleness, or its using method is restricted, crop yield effect after using 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.
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 overcomes the deficiencies in the prior art, provides a kind of preparation method of microbial fertilizer.
A kind of method of producing microbial fertilizer, aspergillus niger culture, phosphorus decomposing bacillus megaterium, plant lactobacillus and subtilis are mixed in proportion as microbial fertilizer, the parts by weight of described microbial fertilizer consist of: aspergillus niger culture 18-26 part, phosphorus decomposing bacillus megaterium 3-5 part, plant lactobacillus agent 10-20 part, bacillus subtilis microbial agent 25-36 part.
Preparation cultivated by bacillus subtilis microbial inoculum: cultivate subtilis from inclined-plane switching, the seed liquor after spreading cultivation step by step is transferred in fermentor tank, obtains bacillus subtilis microbial inoculum after complete fermentation liquor of fermenting separation is dry.
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 0.5-0.7:1, and vehicle group becomes: CaCO
320-40 part, dextrin 10-20 part.Fluidised bed drying, drying temperature 50 DEG C.
Prepared by aspergillus niger culture: spawn culture, and solid fermentation is cultivated: spore liquid is inoculated in solid state fermentation culture material, and 26-35 DEG C is cultured to mycelia and covers with culture material, and low temperature fluidized bed dry, pulverize dry thing.
The bacterial 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 China General Microbiological culture presevation administrative centers.Address: No. 3, No. 1, North Star West Road, Chaoyang District, city of BeiJing, China institute; Postcode: 100101
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.
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.
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.
Industrial at bio-feritlizer, aspergillus niger has cracking larger molecular organics and indissoluble inorganics, is convenient to Crop and utilizes, improve Soil structure, strengthen soil fertility, improve 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 lacking phosphorus in soil, and to raising phosphate fertilizer utilization efficiency, the environmental pollution that the use reducing 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, and pulp delicacy can be made clear and melodious, and fruit juice enriches, good mouthfeel.
Experiment shows, uses this product to improve grain yield 8-30%, improves vegetable crop 10-40%, also can improve vegetables, the quality of grain and local flavor.The amount of every mu of land use fertilizer of the present invention is 50-100 gram, is one of microbial fertilizer that usage quantity is less both at home and abroad at present.In microbial fertilizer of the present invention, add the tap water of 120 times and the urea of 6 times, activation 28 hours in the water temperature of less than 45 DEG C more than 22 DEG C, then together with chemical fertilizer (according to last year usage quantity 50%) be spilled in soil.Preferably make base manure, annual use 1-3 time.
Embodiment
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.
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 product Thermostable α-Amylase is high, heat-resisting, acid resistance is strong.
Thermostable α-Amylase enzyme activity prepared by described bacterial strain is 30000-35000u/ml; Applicable temperature scope is 105-115 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, and the enzyme complete stability alive when pH value is 3.0, 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, 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 produced Thermostable α-Amylase 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.
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%, (NH)
2sO
40.4%, K
2hPO
40.8%, CaCl
20.2%.
Described shake flask culture conditions: this bacterium in the 250mL shaking flask containing 30mL fermention medium, inoculum size 10% (V/V), 100r/min, 40 DEG C of fermentation culture 72h.
Described high-temperature resistant alpha-amylase, its zymologic property is as follows:
(1) this enzyme thermal adaptation a wider range, optimum temperature between 100-110 DEG C, and to be preserved below 110 DEG C, and temperature stability is better, and more than 110 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.
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, and 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, and smooth surface is fine and close, and form is circular, and edge is more neat.Physicochemical characteristics is: catalase (-), gelatine liquefication (-), indoles experiment (+), mobility (-), fermentation gas (-), nitrate reductase (-), and fermentation gas (-) produces hydrogen sulfide (-), grows (+) in pH4.5MRS substratum.
Plant lactobacillus of the present invention adopts following flow process to carry out seed selection:
The original bacterial classification that sets out → test tube activation → ethyl sulfate (DES) mutagenesis → dull and stereotyped primary dcreening operation → nitrosoguanidine (NTG) mutagenesis → dull and stereotyped primary dcreening operation → shaking flask sieves → mitotic stability test again.
The original bacterial classification that sets out is CICC20242, is purchased from Chinese industrial Microbiological Culture Collection 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, DES and NTG is adopted to carry out mutagenesis to this bacterial classification successively, mutagenesis adopts MRS calcium carbonate flat board to carry out primary dcreening operation, then 500mL shake flask fermentation is adopted, biosensor analysis instrument carries out multiple sieve to Producing Strain, the lactobacterium plantarum strain that seed selection is excellent, then does passage assays, 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, therefore using the object bacterial strain that bacterial strain CGMCC No.7928 obtains as seed selection.
Object bacterial strain CGMCC No.7928 is done the experiment of 10L fermentor tank, result shows: after fermentation 72h, take xylan as carbon source, the lactic acid concn of plant lactobacillus CGMCC No.7928 can reach 57g/L, improves 356% compared with starting strain.
Object bacterial strain CGMCC No.7928 is done the experiment of 10L fermentor tank, 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, 200rpm, cultivates about 12h for 40 DEG C, makes thalline be in logarithmic growth in earlier stage.
2) get 5mL bacterium liquid, the centrifugal 10min of 5000rpm collects thalline, with brine 2 times.
3) 107/mL bacteria suspension is diluted to pH7.0 phosphoric acid buffer.
4) get the potassium phosphate buffer of 32mLpH7.0,8mL bacteria suspension, 150mL triangular flask that 0.4mLDES to put into rotor in advance fully mix, make DES ultimate density be 1%(v/v).
5) in 30 DEG C of shaking tables, 150rpm reacts 30min, gets 1mL mixed solution, adds 0.5mL25%Na
2s
2o
3solution stopped reaction.
6) dilution spread screens in solid medium plate in the MRS xylan containing 90g/L xylan.The bacterial strain that after cultivating 2 ~ 3 days at 40 DEG C, picking transparent circle/colony diameter is maximum, 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, 200rpm, cultivates about 12h for 40 DEG C, makes thalline be in logarithmic growth in earlier stage.
2) get the centrifugal 10min of 5mL bacterium liquid 5000rpm and collect thalline, with brine 2 times.
3) 107/mL bacteria suspension is diluted to pH6.0 phosphoric acid buffer.
4) 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.
5) 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.
6) suitably dilute, get last dilution bacterium liquid 0.2mL, dilution spread screens in solid medium plate in the MRS xylan containing 90g/L xylan.The bacterial strain 150 that after cultivating 2 ~ 3 days at 40 DEG C, picking transparent circle/colony diameter is larger.
3. shaking flask is sieved again
1) on super clean bench, get plant lactobacillus one ring on each test tube slant respectively, access is equipped with in the 250mL triangular flask of 50mL liquid MRS xylan substratum, 200rpm, cultivates 3-4 days, detects glucose concn and Pfansteihl change in concentration every day for 40 DEG C.After fermentation ends, compare the xylan wear rate of 150 strain bacterial classifications and lactic acid and produce speed, the transformation efficiency of lactic acid and heteroacid content.
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 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.
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 Laboratories Accession 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 (be called for short CGMCC, address is: No. 3, No. 1, North Star West Road, Chaoyang District, city of BeiJing, China institute, postcode 100101) on July 15th, 2013, and preserving number is CGMCCNO.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: 12OBrix wort l000mL, pH value nature, 121 DEG C of sterilizing 20min;
2) spore suspension preparation (following steps all aseptically operate): add 15mL sterilized water to test tube slant, spore is scraped, with filter paper filtering, filtered solution poured into sterilizing and be added with in the 150mL triangular flask of 5-10 grain sterile glass beads, triangular flask is put into shaking table vibration 10-15min, spore is disperseed.
3) nitrosoguanidine (NTG) mutagenesis
1. with sterilized water spore suspension is adjusted to and is diluted to 106-107/mL.
2. 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.
3. 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.
4. suitably spore concentration is adjusted to 103/mL by dilution, and 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).
5. sieve again: the 200 strain bacterium obtained are inoculated in slant medium with sterile toothpick respectively, and 30 DEG C are cultured to spore and are paved with inclined-plane.Respectively spore is equipped with 50mL and sieves again in the 250mL triangular flask of substratum ferment to be inoculated under aseptic washing, inoculum size 10%(v/v), 30 DEG C, 100r/min cultivates 96h, measures the cellulase activity of each bacterial strain respectively.(the described substratum of sieve is again composed as follows: cellulose powder 50g, ammonium sulfate 5g, magnesium sulfate 0.25g, potassium primary phosphate 1g, sodium-chlor 0.1g, tap water constant volume 1000mL, pH value 5-6,121 DEG C of sterilizing 20min).The bacterial strain choosing cellulose enzyme vigor the highest carries out amplification test.
4) genetic stability test
Li-2013-03 bacterial strain is gone down to posterity for continuous ten times on inclined-plane, and detects the fermentation situation after at every turn going down to posterity by the method that shaking flask is sieved again.Experiment finds, inclined-plane goes down to posterity for continuous ten times, and this bacterial classification proterties does not have considerable change, and property indices is all normal, illustrates that the genetic stability of this bacterial classification is stronger.
5) scale-up
1. seed culture: strains A 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.
2. seed tank culture: by seed liquor with 10%(v/v) inoculum size access be equipped with in the 10L fermentor tank of 7.5L fermented liquid, 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 spergillus 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 Aspergillus niger Li-2010.
Embodiment 2
A kind of microbial fertilizer, parts by weight consist of: aspergillus niger culture 20 parts, phosphorus decomposing bacillus megaterium 4 parts, plant lactobacillus agent 15 parts, bacillus subtilis microbial agent 30 parts.
The preparation method of bacillus subtilis microbial inoculum:
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.Fermented liquid adopts conventional centrifugal freeze-drying method to obtain bacillus subtilis microbial inoculum.
Substratum forms: glucose 6%, yeast extract 1%, peptone 0.2%, CaCO
31%, pH6.8.
The preparation method of plant lactobacillus agent:
(1) first order seed is cultivated: accessed by plant lactobacillus bacterial classification in 500 ml shake flasks, substratum loading amount 100 milliliters, 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, culture temperature 30 DEG C, incubation time 24 hours;
(4) first class seed pot is cultivated: the first class seed pot being 150L with 5% inoculum size access cubic capacity by three grades of seeds, fermention medium loading amount 100L, culture temperature 30 DEG C, tank pressure 0.05MPa, incubation time 18 hours;
(5) fermentor cultivation: it is 3 tons of secondary seed tanks that first class seed pot bacterial classification is accessed cubic capacity with 5% inoculum size, fermention medium loading amount 2 tons, culture condition culture temperature 30 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
2o0.005%, CaCO
32%, agar 1.5%, pH6.8.
The preparation method of aspergillus niger culture:
Slant strains activation culture: be transferred on slant medium by aspergillus niger slant strains, cultivates 3 days for 27 DEG C.
Solid first order seed is cultivated: the access of picking aspergillus niger slant strains is equipped with in 500 milliliters of triangular flasks of 100 grams of substratum and is carried out seed culture, cultivates 3 days for 30 DEG C.
Solid secondary seed is cultivated: stirred by above-mentioned cultured solid first order seed and carry out seed culture, culture condition for adding in 5000 milliliters of triangular flasks that 1000 grams of substratum are housed after fragment: cultivate 3 days for 30 DEG C.
Solid fermentation is cultivated: pulverized by second-level shake flask seed, add in the fermentation vat or pallet that sterilising medium is housed and mix rear cultivation, and bent material culture temperature controls at 26-35 DEG C, humidity 80-90%, every 10 hours stirrings once, and incubation time 5-7 days; The cultivation of solid koji material adopts conventional bent material culture technique; Treat culture material cover with mycelia can terminate cultivate, substratum in advance through high temperature steaming sterilising treatment, sterilising conditions control temperature 121 DEG C, 1 hour time.
Drying and crushing: fermentation ends culture material carries out drying on fluidized-bed or other drying plants, drying temperature controls, at 60 DEG C, to be dried to moisture content below 10%, then to be pulverized by solid culture medium, and crushing material aperture is more than 60 orders.
Substratum forms: solid material: wheat bran 70%, the crop material 15% of pulverizing, soybean cake powder 5%, W-Gum 10%, adds equivalent tap water; Initial pH nature.
Embodiment 3
A kind of microbial fertilizer, parts by weight consist of: aspergillus niger culture 22 parts, phosphorus decomposing bacillus megaterium 3 parts, plant lactobacillus agent 18 parts, bacillus subtilis microbial agent 32 parts.
Produce a method for microbial fertilizer, aspergillus niger culture, phosphorus decomposing Bacillus megaterium, plant lactobacillus and subtilis are combined as microbial fertilizer in proportion.
Preparation cultivated by bacillus subtilis microbial inoculum: cultivate subtilis from inclined-plane switching, the seed liquor after spreading cultivation step by step is transferred in fermentor tank, and the complete fermented liquid that ferments obtains subtilis culture after Plate Filtration, drying.
The preparation 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 0.6:1, and vehicle group becomes: CaCO330 part, 16 parts, dextrin.Fluidised bed drying, drying temperature 50 DEG C.
Prepared by aspergillus niger culture: spawn culture, and solid fermentation is cultivated: spore liquid is inoculated in solid state fermentation culture material, and 30 DEG C are cultured to mycelia and cover with culture material, and low temperature fluidized bed 35 DEG C dry, pulverize dry thing.
Embodiment 4
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 2012.
Experimental plot reaches field maize planting 10 mu, using product every mu of the present invention 0.1 kilogram respectively, emerging about 1 month and using invention product 0.6 kilogram by loose ground mode when planting, and control group uses common fertilizer.
Invention product uses corn field corn yield to reach 680 kilograms, and control group reaches 490 kilograms; This plot was at the 2nd year plant spring wheat, and spring wheat production reaches 410 kilograms, improves 21% than control group per unit area yield.And experimental plot Soil structure is good, without bulk with harden.
Claims (5)
1. a preparation method for microbial fertilizer, comprises the steps: aspergillus niger culture, phosphorus decomposing bacillus megaterium, plant lactobacillus and subtilis to be mixed in proportion as microbial fertilizer;
The parts by weight of described microbial fertilizer consist of: aspergillus niger culture 18-26 part, phosphorus decomposing bacillus megaterium 3-5 part, plant lactobacillus agent 10-20 part, bacillus subtilis microbial agent 25-36 part; Described subtilis preserving number is CGMCCNo.7926, and described aspergillus niger deposit number is CGMCC NO.7927, and described plant lactobacillus deposit number is CGMCC No.7928;
The preparation method of described bacillus subtilis microbial inoculum is as follows: adopt slant strains to spread cultivation step by step and obtain fermentation of bacillus subtilis liquid, and separation of fermentative broth drying obtains;
The preparation method of plant lactobacillus agent is as follows: 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 0.5-0.7:1, and vehicle group becomes: CaCO
320-40 part, dextrin 10-20 part; Fluidised bed drying, drying temperature 50 DEG C;
The preparation method of described aspergillus niger culture is as follows: solid fermentation is cultivated end culture material and carry out drying on fluidized-bed or other drying plants, drying temperature controls at 60 DEG C, be dried to moisture content below 10%, then pulverized by solid culture medium, crushing material aperture is more than 60 orders.
2. the preparation method of a kind of microbial fertilizer according to claim 1, it is characterized in that, microbial fertilizer parts by weight consist of: aspergillus niger culture 20 parts, phosphorus decomposing bacillus megaterium 4 parts, plant lactobacillus agent 15 parts, bacillus subtilis microbial agent 30 parts.
3. the preparation method of a kind of microbial fertilizer according to claim 2, is characterized in that, the preparation method of bacillus subtilis microbial inoculum 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; Fermented liquid adopts conventional centrifugal freeze-drying method to obtain bacillus subtilis microbial inoculum;
Substratum forms: glucose 6%, yeast extract 1%, peptone 0.2%, CaCO
31%, pH6.8;
The preparation method of plant lactobacillus agent is as follows:
(1) first order seed is cultivated: accessed by plant lactobacillus bacterial classification in 500 ml shake flasks, substratum loading amount 100 milliliters, 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, culture temperature 30 DEG C, incubation time 24 hours;
(4) first class seed pot is cultivated: the first class seed pot being 150L with 5% inoculum size access cubic capacity by three grades of seeds, fermention medium loading amount 100L, culture temperature 30 DEG C, tank pressure 0.05MPa, incubation time 18 hours;
(5) fermentor cultivation: it is 3 tons of secondary seed tanks that first class seed pot bacterial classification is accessed cubic capacity with 5% inoculum size, fermention medium loading amount 2 tons, culture condition culture temperature 30 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
2o0.005%, CaCO
32%, agar 1.5%, pH6.8;
The preparation method of aspergillus niger culture is as follows:
Slant strains activation culture: be transferred on slant medium by aspergillus niger slant strains, cultivates 3 days for 27 DEG C;
Solid first order seed is cultivated: the access of picking aspergillus niger slant strains is equipped with in 500 milliliters of triangular flasks of 100 grams of substratum and is carried out seed culture, cultivates 3 days for 30 DEG C;
Solid secondary seed is cultivated: stirred by above-mentioned cultured solid first order seed and carry out seed culture, culture condition for adding in 5000 milliliters of triangular flasks that 1000 grams of substratum are housed after fragment: cultivate 3 days for 30 DEG C;
Solid fermentation is cultivated: pulverized by second-level shake flask seed, add in the fermentation vat or pallet that sterilising medium is housed and mix rear cultivation, and bent material culture temperature controls at 26-35 DEG C, humidity 80-90%, every 10 hours stirrings once, and incubation time 5-7 days; The cultivation of solid koji material adopts conventional bent material culture technique; Treat culture material cover with mycelia can terminate cultivate, substratum in advance through high temperature steaming sterilising treatment, sterilising conditions control temperature 121 DEG C, 1 hour time;
Drying and crushing: fermentation ends culture material carries out drying on fluidized-bed or other drying plants, drying temperature controls, at 60 DEG C, to be dried to moisture content below 10%, then to be pulverized by solid culture medium, and crushing material aperture is more than 60 orders;
Substratum forms: solid material: wheat bran 70%, the crop material 15% of pulverizing, soybean cake powder 5%, W-Gum 10%, adds equivalent tap water; Initial pH nature.
4. the preparation method of a kind of microbial fertilizer according to claim 1, it is characterized in that, microbial fertilizer parts by weight consist of: aspergillus niger culture 22 parts, phosphorus decomposing bacillus megaterium 3 parts, plant lactobacillus agent 18 parts, bacillus subtilis microbial agent 32 parts.
5. the preparation method of a kind of microbial fertilizer according to claim 4, it is characterized in that, the preparation method of plant lactobacillus agent is as follows: from inclined-plane switching culturing plants Bacterium lacticum, seed liquor after spreading cultivation step by step is transferred in fermentor tank, 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 0.6:1, and vehicle group becomes: CaCO330 part, 16 parts, dextrin; Fluidised bed drying, drying temperature 50 DEG C.
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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 |
| CN109652336A (en) * | 2019-01-22 | 2019-04-19 | 广西金穗生态科技股份有限公司 | One plant of Bei Laisi bacillus and its application |
| CN111996122A (en) * | 2020-08-27 | 2020-11-27 | 甘肃省科学院生物研究所 | Preparation process of low-cost solid microbial inoculum |
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| CN102212494A (en) * | 2011-04-12 | 2011-10-12 | 王颖 | Organic matter decomposing inoculant, and preparation method and application thereof |
| 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 |
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| WO2012083346A1 (en) * | 2010-12-23 | 2012-06-28 | Ams Laboratories Pty Ltd | Inoculum and method of preparation |
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