CN103739322B - A kind of preparation method of composite bio-fertilizer - Google Patents
A kind of preparation method of composite bio-fertilizer Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 29
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- 241000894006 Bacteria Species 0.000 claims abstract description 57
- 241000186660 Lactobacillus Species 0.000 claims abstract description 24
- 229940039696 lactobacillus Drugs 0.000 claims abstract description 24
- 239000000463 material Substances 0.000 claims abstract description 18
- 241001513093 Aspergillus awamori Species 0.000 claims abstract description 16
- 230000003381 solubilizing effect Effects 0.000 claims abstract description 16
- 229910052700 potassium Inorganic materials 0.000 claims abstract description 15
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 14
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims abstract description 11
- 239000000084 colloidal system Substances 0.000 claims abstract description 11
- 239000011591 potassium Substances 0.000 claims abstract description 11
- 239000002068 microbial inoculum Substances 0.000 claims abstract description 9
- 229910019142 PO4 Inorganic materials 0.000 claims abstract description 8
- 239000010452 phosphate Substances 0.000 claims abstract description 8
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims abstract description 8
- 238000002156 mixing Methods 0.000 claims abstract description 5
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- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Natural products OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 2
- 239000000417 fungicide Substances 0.000 description 2
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- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 2
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 2
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 2
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/141—Feedstock
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Fertilizers (AREA)
Abstract
The invention discloses a kind of preparation method of composite bio-fertilizer, belong to agricultural fertilizer technical field.The preparation method of described composite bio-fertilizer comprises and being consisted of by parts by weight: colloid bud pole bacterium 5-18 part, phosphate solubilizing bacteria 2-6 part, potassium solubilizing bacteria 5-10 part, subtilis culture 18-30 part, Aspergillus awamori culture 5-10 part, the microbial inoculum mixing of plant lactobacillus agent 3-8 part, or granulate after mixing.Fertilizer of the present invention can strengthen the drought-resistant ability of crop, and the colloid bud pole bacterium in composite bacteria of the present invention is the silicate bacteria utilizing the ability of dissolving potassium that screens from crop root very strong, adopts specific substratum, develops through industrial fermentation; This bacterium can secrete the growth-promoting substance such as growth hormone material and Plant hormones regulators,gibberellins material, extraneous root strong sprout; Decomposing element silicon in soil, for plant utilization, makes the wax layer of plant thicken, and improves plant water keeping ability; Can promote that soil aggregate is formed, and keeps soil from packing together; Spoiled soil capillarity, prevents soil water evaporation.
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 composite bio-fertilizer.
Background technology
Pollution by chemical fertilizer has become the large public hazards in the world today one.Agricultural experts unanimously appeal, Environment control, must reduce fertilizer amount.Expert, the new academician of China Agricultural University Chen Wen think, the excessive chemical fertilizer of using of current China arrives the limit.For nitrogenous fertilizer, the average amount of application of China is nearly 3 times, more than 8 times of Australia of the U.S., which results in that Soil structure is destroyed, a series of serious problems such as nature biotechnology diversity and ecosystem stability reduction.Agricultural microbiology Professional Committee of Chinese Society for Microbiology director doctor Li Jun points out, widelys popularize microorganism fertilizer and coordinates chemical fertilizer application to be exactly one of solution to this problem.
Microorganism fertilizer a kind ofly causes farm crop to obtain the living microorganisms goods of specific fertilizer effect with microbial life activity and product thereof, it at culture fertility, improve chemical fertilizer utilization ratio, suppress the absorption of the objectionable impurities such as farm crop heavy metal and agricultural chemicals, have irreplaceable effect in utilizations of becoming thoroughly decomposed, raising quality of agricultural product etc. of purification and rehabilitating soil, promotion agricultural crop straw and municipal wastes.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 chemical fertilizer, fertilizer, the multiple fertility of beneficial organism bacterium and effect, is activated fertilizer nutrient, improves effect of fertilizer, Crop Improvement quality, promotes the desirable fertilizer of agricultural production efficiency.Microbial fertilizer is live body fertilizer, and a large amount of beneficial microorganism vital movements that its effect mainly contains by it have been come.Only have under these beneficial microorganisms are in vigorous breeding and metabolic situation, Substance Transformation and beneficial metabolic product could constantly be formed.Therefore, whether beneficial microorganism kind in microbial fertilizer, vital movement vigorous is the basis of its validity, and unlike other fertilizer be by the form of the principal elements such as nitrogen, phosphorus, potassium and how many based on.Just because of microbial fertilizer is preparation of living, thus its fertilizer efficiency and number of viable, intensity and ambient environmental conditions closely related, comprise temperature, moisture, potential of hydrogen, nutritional condition and formerly live in indigenous microorganism repulsive interaction in soil and have certain influence.
For many years, most processing modes of microbial fertilizer only adopt finished product microbial inoculum to mix with filler, lack fermentating metabolism product, have a strong impact on the fertilizer efficiency of microbial fertilizer product, such as publication No. is the patent application of CN103396252A, disclose a kind of containing amino acid whose composite microbiological fertilizer, the finished product microbial inoculum wherein added is mixed according to a certain percentage by subtilis, Bacillus licheniformis and bacillus megaterium and forms.
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 CN102888356A, 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.
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 composite bio-fertilizer.
The preparation method of described composite bio-fertilizer is as follows: the microbial inoculum mixing formed according to following parts by weight, or granulates after mixing:
Colloid bacillus cereus 5-18 part, phosphate solubilizing bacteria 2-6 part, potassium solubilizing bacteria 5-10 part, subtilis culture 18-30 part, Aspergillus awamori culture 5-10 part, plant lactobacillus agent 3-8 part;
Described subtilis culture preparation: 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 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 0.5-0.6:1, and vehicle group becomes: CaCO320-30 part, dextrin 10-15 part.Fluidised bed drying, drying temperature 50 DEG C.
Prepared by Aspergillus awamori culture: spawn culture, and solid fermentation is cultivated: spore liquid is inoculated in aspergillus oryzae solid state fermentation culture material, and 26-33 DEG C is cultured to mycelia and covers with culture material, and low temperature fluidized bed dry, pulverize dry thing.
The bacterial classification that the present invention adopts is as follows:
Subtilis (Bacillussubtilissubsp) CGMCC7926
Plant lactobacillus (Lactobacillusplantarum) CGMCC7928
Aspergillus awamori (Aspergillusawamori) CGMCC3.6484
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.
Phosphate solubilizing bacteria is phosphorus decomposing bacillus megaterium, and phosphorus decomposing microbial inoculum is provided by Cangzhou prosperous generation thing technical institute, address: liberation West Road, Hebei China Cangzhou City Canal Zone chin or cheek and Building A 1, international business affairs center district 807-812.
Potassium solubilizing bacteria is provided by infinitesimal bio tech ltd, Guangzhou, address: No. 101 506, science main road, Luogang District, Guangzhou.
Rui Gu bio tech ltd, Baoding provides colloid bacillus cereus bacterium powder.
Beneficial effect
Fertilizer of the present invention can strengthen the drought-resistant ability of crop.Colloid bacillus cereus in composite bacteria of the present invention is the silicate bacteria that ability of dissolving potassium is very strong, adopts specific substratum, develops through industrial fermentation.This bacterium can secrete the growth-promoting substance such as growth hormone material and Plant hormones regulators,gibberellins material, extraneous root strong sprout; Decomposing element silicon in soil, for plant utilization, makes the wax layer of plant thicken, and improves plant water keeping ability; Can promote that soil aggregate is formed, and keeps soil from packing together; Spoiled soil capillarity, prevents soil water evaporation.
Fertilizer of the present invention can improve soil.In fertilizer, beneficial microorganism can produce glucide and plant mucilage, mineral idiosome and organic colloid combine, and can improve soil aggregate, strengthen the physicals of soil and reduce the loss of soil particle, under certain conditions, soil ulmin can also be participated in formed.So use microbial fertilizer can improve soil physical property, be conducive to increasing soil fertility.
This product using method is simple, every mu of ground usage quantity 0.3-1 kilogram, and cost is only 80-100 unit/mu, and seed dressing or front earth's surface of pouring water vegetative period are sprayed.
The conversion of soil organisms enzyme, reducing agriculture production cost, reducing the use of chemical fertilizer, recovering all can play significant effect in soil ecology soil fertility and effective raising crop yield etc., gives full play to the organic usefulness of soil.
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 (Bacillussubtilis) 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 CGMCCNo.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 (Bacillussubtilis) 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,
Be 1 enzyme activity unit, represent 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 (Lactobacillusplantarum) Li-2013-01, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCCNo.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 (-), fermentation gas (-), produce hydrogen sulfide (-), in pH4.5MRS substratum, grow (+).
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 CGMCCNo.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 CGMCCNo.7928 obtains as seed selection.
Object bacterial strain CGMCCNo.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 CGMCCNo.7928 can reach 57g/L, improves 356% compared with starting strain.
Object bacterial strain CGMCCNo.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 CGMCCNo.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.
The preparation method of Aspergillus awamori microbial inoculum:
Slant strains activation culture: be transferred on slant medium by Aspergillus awamori slant strains, cultivates 3 days for 27 DEG C.
Solid first order seed is cultivated: the access of picking Aspergillus awamori 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 80, soybean cake powder 10%, W-Gum 10%, adds equivalent tap water; Initial pH nature.
Embodiment 2
A kind of composite bio-fertilizer, parts by weight consist of: colloid bacillus cereus 12 parts, phosphate solubilizing bacteria 5 parts, potassium solubilizing bacteria 8 parts, subtilis culture 25 parts, Aspergillus awamori culture 7 parts, plant lactobacillus agent 6 parts.
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 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.The complete fermented liquid that ferments obtains containing bacillus subtilis microbial inoculum at interior subtilis culture after Plate Filtration, drying.
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.
Embodiment 3
A kind of composite bio-fertilizer, parts by weight consist of: colloid bacillus cereus 10 parts, phosphate solubilizing bacteria 6 parts, potassium solubilizing bacteria 7 parts, subtilis culture 27 parts, Aspergillus awamori culture 9 parts, plant lactobacillus agent 7 parts.
Described subtilis culture preparation: 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 method of plant lactobacillus agent: from inclined-plane switching culturing plants Bacterium lacticum, the seed liquor after spreading cultivation step by step is transferred in fermentor tank, and the complete fermentation liquor low-temperature negative-pressure that ferments is concentrated in vacuo to 45% of original volume, obtains bacterium concentrated solution.Add carrier: in concentrated solution, add the carrier mixed, mix; The weight ratio of concentrated solution and carrier is 0.5:1, and vehicle group becomes: CaCO
325 parts, 12 parts, dextrin.Fluidised bed drying, drying temperature 50 DEG C.
Prepared by Aspergillus awamori culture: spawn culture, and solid fermentation is cultivated: spore liquid is inoculated in aspergillus oryzae solid state fermentation culture material, and 27-33 DEG C is cultured to mycelia and covers with culture material, and low temperature fluidized bed 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-September 30 April in 2009.
Experimental plot reaches field maize planting 10 mu, using product every mu of the present invention 0.5 kilogram respectively, emerging about 1 month and using invention product 0.5 kilogram by loose ground mode when planting, and control group uses common fertilizer.
Invention product uses corn field corn yield to reach 650 kilograms, and control group reaches 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 reaches 400 kilograms, improves 20% 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 composite bio-fertilizer, the microbial inoculum formed according to following parts by weight mixes, or granulates after mixing:
Colloid bacillus cereus 5-18 part, phosphate solubilizing bacteria 2-6 part, potassium solubilizing bacteria 5-10 part, subtilis culture 18-30 part, Aspergillus awamori culture 5-10 part, plant lactobacillus agent 3-8 part; Described subtilis preserving number is CGMCCNo.7926, and described plant lactobacillus deposit number is CGMCCNo.7928;
The preparation method of described subtilis culture is as follows: the acquisition of fermented liquid: cultivate subtilis from inclined-plane switching, seed liquor after spreading cultivation step by step is transferred in fermentor tank, and the complete fermented liquid that ferments obtains subtilis culture after Plate Filtration, drying;
The preparation method of described 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 0.5-0.6:1, and vehicle group becomes: CaCO
320-30 part, dextrin 10-15 part; Fluidised bed drying, drying temperature 50 DEG C;
The preparation method of described Aspergillus awamori culture is as follows: spawn culture, and solid fermentation is cultivated: spore liquid is inoculated in aspergillus oryzae solid state fermentation culture material, and 26-33 DEG C is cultured to mycelia and covers with culture material, and low temperature fluidized bed dry, pulverize dry thing.
2. the preparation method of a kind of composite bio-fertilizer according to claim 1, is characterized in that, composite bio-fertilizer parts by weight consist of: colloid bacillus cereus 12 parts, phosphate solubilizing bacteria 5 parts, potassium solubilizing bacteria 8 parts, subtilis culture 25 parts, Aspergillus awamori culture 7 parts, plant lactobacillus agent 6 parts.
3. the preparation method of a kind of composite bio-fertilizer according to claim 2, is characterized in that, the preparation method of subtilis culture is as follows:
The acquisition of fermented liquid: adopt slant strains to spread cultivation step by step and obtain fermentation of bacillus subtilis liquid;
(1) first order seed is cultivated: subtilis slant strains accessed in 500 ml shake flasks, substratum loading amount 100 milliliters, rotary shaker 180 revs/min, culture temperature 30 DEG C, incubation time 24 hours;
(2) secondary seed cultivate: by first order seed according to 10% inoculum size access in 500 milliliters of secondary seed shaking flasks, culture condition is identical with first order seed;
(3) three grades of seed culture: secondary seed is accessed in 5000 milliliters of three grades of seed flask with 10% inoculum size, substratum loading amount 1000 milliliters, rotary shaker 100 revs/min, culture temperature 30 DEG C, incubation time 24 hours;
(4) first class seed pot is cultivated: the first class seed pot being 150L with 10% inoculum size access cubic capacity by three grades of seeds, fermention medium loading amount 100L, culture temperature 28 DEG C, stirring velocity 100 revs/min, ventilation (V/V) 1:0.5, tank pressure 0.05MPa, incubation time 24 hours;
(5) fermentation culture: it is 1.5 tons of secondary seed tanks that first class seed pot bacterial classification is accessed cubic capacity with 10% inoculum size, fermention medium loading amount 1 ton, culture condition culture temperature 28 DEG C, stirring velocity 100 revs/min, ventilation (V/V) 1:0.5, tank pressure 0.05MPa, incubation time 24 hours; The complete fermented liquid that ferments obtains containing bacillus subtilis microbial inoculum at interior subtilis culture after Plate Filtration, drying;
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%, dibasic ammonium citrate 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.
4. the preparation method of a kind of composite bio-fertilizer according to claim 1, is characterized in that, composite bio-fertilizer parts by weight consist of: colloid bacillus cereus 10 parts, phosphate solubilizing bacteria 6 parts, potassium solubilizing bacteria 7 parts, subtilis culture 27 parts, Aspergillus awamori culture 9 parts, plant lactobacillus agent 7 parts.
5. the preparation method of a kind of composite bio-fertilizer according to claim 4, it is characterized in that, described subtilis culture preparation: cultivate subtilis from inclined-plane switching, seed liquor after spreading cultivation step by step is transferred in fermentor tank, and the complete fermented liquid that ferments obtains subtilis culture after Plate Filtration, drying;
The preparation method of plant lactobacillus agent: from inclined-plane switching culturing plants Bacterium lacticum, the seed liquor after spreading cultivation step by step is transferred in fermentor tank, and the complete fermentation liquor low-temperature negative-pressure that ferments is concentrated in vacuo to 45% of original volume, obtains bacterium concentrated solution; Add carrier: in concentrated solution, add the carrier mixed, mix; The weight ratio of concentrated solution and carrier is 0.5:1, and vehicle group becomes: CaCO
325 parts, 12 parts, dextrin; Fluidised bed drying, drying temperature 50 DEG C;
Prepared by Aspergillus awamori culture: spawn culture, and solid fermentation is cultivated: spore liquid is inoculated in aspergillus oryzae solid state fermentation culture material, and 27-33 DEG C is cultured to mycelia and covers with culture material, and low temperature fluidized bed dry, pulverize dry thing.
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